1

Appendix C– Open Literature Review of Amphibian Data 

(248 pages) 

A table that summarizes the reported results for each available amphibian study along with 

selected test design elements and identified uncertainties or concern are presented in the table 

below. A summary of the studies along with the individual reviews for the available studies (in 

alphabetic order) since the 2007 SAP are provided after the table. 

Table of Contents

1. Table A. Summary of Available Amphibians Open Literature Data (that passed the 

ECOTOX and OPP screen).............................................................................................. 3

2. Summary of Amphibian Open Literature as of the 2007 SAP on Amphibians ........ 22

3. INDIVIDUAL OPEN LITERATURE SUMMARIES (in alphabetic order) ............ 40

3.1. Allran, J.W., W.H. Karasov. 2000 ................................................................................ 40

3.2. Allran J.W and W.H. Karasov. 2001 ......................................................................44

3.3. Birge, W. J., J. A. Black and R. A. Kuehne. 1980 ...................................................45

3.4. Boone, M.D. and C.M. Bridges-Britton. 2006 ........................................................47

3.5. Boone, M. D. and S. M. James. 2003 ........................................................................49

3.6. Brodeur, J.C., G. Svartz, C.S. Perez-Coll, D.J.G. Marino, J. Herkovits 2009. ....57

3.7. Brodkin, M.A., H. Madhoun, M. Rameswaran, and I. Vatnick. 2007. ................63

3.8. Choung, C.B, R.V. Hyne, R.M. Mann, M.M. Stevens, G.C. Hose. 2011. .............66

3.9. Clements, C., S. Ralph, and M. Petras. 1997. .........................................................69

3.10. Diana, S. G., Resetarits, W. J. Jr., Schaeffer, D. J., Beckmen, K. B., and 

Beasley, V. R. (2000). .................................................................................................71

3.11. Du Preez, L. H., N. Kunene, G. J. Everson, J. A. Carr, J. P. Giesy, T. S. 

Gross, A.J. Hosmer, R. J. Kendall, E.E. Smith, K. R. Solomon, G.J. Van Der 

Kraak. 2008. ..............................................................................................................74

3.12. Forson, D. and Storfer, A. (2006). ............................................................................76

3.13. Forson, D. and Storfer, A. (2006). ............................................................................78

3.14. Fort, D. J., R. L. Rogers, J.H. Thomas, B.O. Buzzard, A. M. Noll and C.D. 

Spaulding. 2004 .........................................................................................................80

3.15. Freeman J.J. and A.L. Rayburn. 2004 ....................................................................81

3.16. Freeman J.L. N. Beccue and A.L. Rayburn. 2005 ..................................................82

3.17. Gibble, R.E. and K.N. Baer. 2011 .............................................................................84

3.18. Gunderson, M.P, N. Veldhoen, R.C. Skirrow, M.K. Macnab, W. Ding, G. 

van Aggelen, and C.C. Helbing. 2011 ......................................................................87

3.19. Hayes, T. B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. 

Adame, E. Chan, D. Bucholz, T. Stueve, and S. Gallipeau. 2010 .........................90

3.20. Haridon J.L., M. Fernandez, V. Ferrier, and J. Bellan. 1993 ...............................99

3.21. Houck, A., and S.K. Sessions. 2006........................................................................101

3.22. Howe, G.E., R. Gillis, and R.C. Mowbray (1998). ................................................105

3.23. Kerby, J.L and A. Storfer. 2009 .............................................................................109

3.24. Kiesecker J.M. 2002 ................................................................................................112

3.25. Kloas, W., I. Lutz, R. Urbatzka, T. Springer, H. Hrueger, J. Wolf, L. 

Holden, and A. Hosmer. 2009 .................................................................................115
2

3.26. Kloas, W., I. Lutz, R. Urbatzka, T. Springer, H. Hrueger, J. Wolf, L. 

Holden, and A. Hosmer. 2009 .................................................................................117

3.28. Koprivnikar, J., M. R. Forbes, and R. L. Baker. 2007 ........................................118

3.29. Koprivnikar, J. 2010 ...............................................................................................120

3.30. LaFiandra, E.M, K.J. Babbitt, and S.A. Sower. 2008 .........................................126

3.31. Langerveld, A.J., R. Celestine, R. Zaya, D. Mihalko, and C.F. Ide. 2009. ........129

3.32. Langlois, V.S., A.C. Carew, B.D. Pauli, M.G. Wade, G.M. Cooke, V.L. 

Trudeau. 2010 ..........................................................................................................132

3.33. Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and Hamilton, 

S. J. (1998) .................................................................................................................137

3.34. Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin. 2008. .......139

3.35. Lenkowski, J.R. and K. A. McLaughlin. 2010. .....................................................141

3.36. Lenkowski, J.R, G.Sanchez-Bravo, K.A. McLaughlin. 2010. .............................145

3.37. Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin. 2008. .......147

3.38. Oka, T., O. Tooi, N. Mitsui, M. Miyahara, Y. Ohnishi, M. Takase, A. 

Kashiwagi, T. Shinkai, N. Santo, T. Iguchi. 2008 .................................................149

3.39. Olivier, H. M. and R. R. Moon. 2010. ...................................................................152

3.40. Orton F., I. Lutz, W. Kloas, and E.J. Routledge. 2009 .........................................156

3.41. Relyea, R. A. 2009 ....................................................................................................157

3.42. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. 

Niedzwiecki, T. Sager, A. Sih, and B.D. Palmer. 2003 ........................................169

3.43. Rohr, J.R. and B.D. Palmer. 2005 .........................................................................171

3.44. Rohr, J.R., T. Sager, T.M. Sesterhenn, and B.D. Palmer. 2006 .........................175

3.45. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. 

Niedzwiecki, T. Sagar, A. Sih, and B.D. Palmer. 2004 ........................................177

3.46. Rohr, J. R. and P. W. Crumrine. 2005 ...................................................................180

3.47. Rohr, J. R., A. M. Schotthoefer, T. R. Raffel, H. J. Carrick, N. Halstead, J. 

T. Hoverman, C. M. Johnson, L. B. Johnson, C. Lieske, M. D. Piwoni, P. K. 

Schoff, and V. R. Beasley. 2008...............................................................................193

3.48. Rohr, J. R., A. Swan, T.R. Raffel, and P.J. Hudson. 2009. .................................206

3.49. Rohr, J. R., T. M. Sesterhenn and C. Stieha. 2011. .............................................209

3.50. Spolyarich, N., R. Hyne, S. Wilson, C. Palmer and M. Byrne. 2010. .................214

3.51. Storrs, S.I. and J.M. Kiesecker. 2004 ....................................................................219

3.52. Storrs Méndez, S.I., D.E. Tillitt, T.A.G Rittenhouse, R.D. Semlitsch. 2009 ......221

3.53. Storrs-Méndez, S. I. and R. D. Semlitsch. 2009 ....................................................224

3.54. Wan M.T., C. Buday, G. Schroeder, J. Kuo, and J. Pasternak. 2006 ................230

3.55. Williams, B. K. and R. D. Semlitsch. 2010 .............................................................232

3.56. Zaya, R. M., Z. Amini, A. S. Whitaker, S. Kohler and C. F. Ide. 2011 ...............236

3.57. Zaya, R. M., Z. Amini, A. S. Whitaker, and C. F. Ide. 2011 ...............................242

4. References from the 2003 and 2007 SAPs on Amphibians ...................................... 247
3

1. Table A. Summary of Available Amphibians Open Literature Data (that passed the 

ECOTOX and OPP screen) 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/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 

Metamorpsubset (50%ability to ginformatiotreatment greported (oreplicates cQualitativeand metamsampling afactors and treatment s

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) Gro (A) Bio/Mol (A) Surv 

2590 20000 20000 2590 20000 20000 20000 

4320 >20000 >20000 4320 >20000 >20000 >20000 

Deformities (tail, facial edema)(E) Mass (E) Swimming (E) Buccal ventilation Mass (A) Hemoglobin Hatch and mortality 

Atrazine mwater qualstate conduQualitativelevels of atsite 

Birge et al. 1980 / E6187 

R. catesbeiana, R. pipiens, R. palustris, B. 

F / 

80 

NS / (Y 

unsure if 

NC or 

glass 

50 -103 E 

/ 0.5L; 2.5 

h 

Surv 4 dph LC50 

-- -- R. catesbeiana = 410 

R. pipiens = 7680 R. palustris = 17960 

LC50s werabnormalitsurvival un

1 TP = tadpole; LV = larvae; M= metamorphorsis; E= embryo; G = gosner stage; NF=Nieuwkoop and Faber; SVL=snout-vent-length; A = 

juvenile; if reported in study, developmental stage or some other identifier at test initiation is given, if not reported than designated as N

2 TS = Test substance; T = technical; F = formulation 3 NA = not applicable; NS = not specified; ACTN = acetone; EtOH= ethanol; DMSO=dimethylsulfoxide; MeOH=methanol; IPA = isop4 Negative Control (NC) and/or Solvent Control (SC) included in study design; MA in CNT = Measurable atrazine in control treatment 

(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 was repo

larvae/tadpoles in a certain test vessel size but test solution volume was not reported, then this is presented # organisms in tank (volume 

actual loading could not be determined; PE = polyethylene 

6 Water 7 Metam = metamorphosis; Gro = growth; Imm/Inf = immune system or infection; Bio/Mol = biochemical/Molecular; Sexdev = sexual 

Behav=behavioral; Surv = survival; N = nominal conc.; M = measured conc; NDR = not dose responsive (no significance at higher dos
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

americanus / NS (purchased) / E for 4 dph 

SC); NS B. americanus >48000 was not id

in source oQualitative 

LC50 valuabnormal e

Brodeur et al. 2009 / E112588 

R. arernarum / LP8/ TP 

(G38/39)-M 

T/ 

98 

NA / 

(Y/NA9); 

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 qmetamorphsign. at 10endpoint (testing for using EC5Qualitativehigher loadreported 

Brodkin et al 2007 / E103351 

R. pipiens / Field (ANA10) / 

A for 8 d 

T/ 

NS 

NA / (N & 

N11/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 froguncertainty treatmentsquantitativof growth, Invalid bas(dilution wused) 

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 refood and wspecified; Qualitativethere is a lwater and 

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 

Limited retest design water solub

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) w10 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 stimu
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

 -- from that conc. Qualitative 

lack of test reported 

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 responsiveInvalid bascontrol mo

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 

25 25 0.1 25 

>25 >25 1 >25 

Mass; SVL Gonad development; laryngeal muscle ↓ estradiol males % mortality 

Higher loa72 hr may delayed mdifference factor); lac(estradiol) Invalid bas

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 

18.4 (M) 18.4 184 

184 184 >184 

↑ larval period ↓ mass & SVL % mortality 

Limited reQualitativeof reported 

higher than water qual

periodicall

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 redose respostress from Qualitativeslightly higlimited wa

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. laevisLC50 = 24500 or 25600 

EC50 = 4100 or 4400 X. tropicalis LC50 = 19400 – 31800 EC50 = 2100 - 9300 

Limited wrecommenallowed unInvalid bas

Freeman & Rayburn 2004 / 

B. americanus & X. laevis / 

T / 

NS 

NS / (C 

used NS if NS NS (2 TP/3L Bio/Mol Surv 800 NS >800 NS Nuclei (represent genotox) Not specified; based on reported ‘N’ Stats for nu

sample siz
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

E78644 Field 

(parents)(Bufo) & Nasco / TP (NF NS) for 3-5 wks 

NC, SC); 

NS 

tank); 

weekly 

in tables appears 100% survival unknown (

Qualitativeuncertainty with no reprenewal pe

Freeman & Rayburn 2005 / E81459 

X. laevis / Nasco/Xenopus Express / TP (varied NF40 to 54) for 3-5 wks 

T / 

98 

NA / 

(Y/NA); 

NS12

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 fluncertainty accounting volume of water qualQualitativemolecular other reasoInvalid for factor; Quastage; howcontrol 

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 quavolume of verify loadresponsive test differe800 in othe

QualitativeMetamorpdefined anweekly renpotential c

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 

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 mpotentially survival noorganisms) appears frofor peptide formulatio

12 Study reports that solutions measured from experimental tanks and atrazine concentrations were within expected range; however uns

control 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

3X/wk 

Invalid for based on lacontaminaconfidenceatrazine foreporting a

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 quapositive codevelopmeInvalid baspositive codevelopme

Gunderson et al2011 / 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 win CYP19 of CYP19 combining Invalid basrecommenused in stu

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 whigher than the control psueorepliQualitative 

solvent chorecommenalso uncertadd to unc

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 wvessels; inreport resusurvival noQualitative higher loadand a renew

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

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

Hayes et al. 2003 

R. pipiens / Field / (48hr post hatch)-M 

T / 

98 

EtOH 

(0.0036) / 

(N/Y) 

NS14

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 statisticdevelopmemetamorphresults not Qualitative higher loadand a renew

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 oand clear dparameters Invalid due to atrazine 

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 quaresults are report; authsignificancQualitative higher loadand a renewinconsistenuncertainty 

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, account fotreatment/r(e.g., # of pcompared metamorphQualitative higher loadand a renewuncertainty uncertaintymetamorph

Hecker et al 2005a / E79287 

X.laevis / Xenopus Express / A for 

T/ 

97.1 

NA 

(Y/NA); 

N 

NS 

1 

frog/10L; 

3 days 

Sexdev Bio/Mol Surv 

258(M) 24.6 -- 

>258 258 -- 

Gondal abnormalities ↓ Plasma testosterone 4 total mortalities (65 exposed) 

Uncertaintcascading not reporte

14 Study indicates concentrations verified; however, unclear if this included controls 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

36 d reported; mortality nor presented in 

terms of NOAEC/LOAEC 

sample sizQualitativeuncertainty evaluation 

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 water qualnitrites, anaromatase Qualitative higher loadand a renew

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 suabove wateQualitative lack of conand testing 

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 antibodysecreting cells (4 wks) Number of Lymphocytes 1 mortality (believed 24 exposed) reported; mortality not presented in terms of NOAEC/LOAEC 

No informand limited parametersor not as teuncertainty to not be femeasuremuncertainty study Invalid basoverall lacparameters 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 

Uncertaintviral load tquality repQualitativehigher than limited warenewal pe

Kiesecker 2002 / E68326 

R. sylvatica /Field /TP 

T / 

NS 

ACNT & 

DMSO PE 1 TP/3.5L; 48 hr Imm/inf Surv <3 NS 3 NS 

↓ eosinophils ↑ trematode infections 

Mortality not reported; based on 

Uncertain exposed to 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

(G25) for 4 wk (NS); 

(Y,Y); NS

sample size in Figure 2 in report survival appears to be 100% 

uncertainty 

scenario; dto cercariareported; uInvalid bascontrol, alsdifferent eparasite, ununcertainty 

Kloas 2009a / E112914 

X. laevis / Xenopus I / TP (8 dpf) for 83 d 

T / 

NS 

NA / 

(Y/NA); 

Y15

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>10016

Sex ratio; gonadal development % Survival 

Atrazine cfluctuationhistologicarelative to Quantitativdeficiencierobust eno

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 bindependentank that climited watest, test sothe control Qualitative lack test de

Koprivnikar et al2007 / E103348 

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

F / 

40.8 

NA / 

(N17/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 sutreatment (variability when tadpatrazine –ulimited waInvalid bascontrol, lacadverse eff

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 of the tw

effect on wt and SVL reported in other study; EFED does not consider the differences in wt and SVL to be a concentration-dependent e

17 A dilution water only control was not included. A control that contained no atrazine but cercariae that were also not exposed to atraz
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

cercariae, wrelevance) 

Koprivnikar et al2010 / E153870 

R. pipiens / E from CBSC18 / 

TP (<G27)- ~ G4319

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 groups; unaccounted reported reorganisms Invalid basbased on Fwater qualand variabgroups add 

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; SUL20

Sex ratio; gonad development % Survival 

Limited wadults used the field or Qualitativeloading wareported wperiod(50% potential e

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 wregarding ohowever, sppb when not be unechanges Invalid fomortality cconcentratlimited wa>48 hours; 

survival band renew

Larson et al. 1998 / E60632 

A. tigrinum / Field / LV 

T / 

NS 

ACTN 

(NS)/ PE 1 TP/4L; 50% at Metam Gro <81.8(M 81.8 81.8 >81.8 ↓metamorphosis Atrazine m

oxygen rep

18 CBSC = Carolina Biological Supply Company (Burlington, NC) 19 G43- first observance of reabsorbed tail 20 SUL = snout-urostyle length 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

(SVL= 30mm) for 86 d 

(N/Y); Y 48hr Bio/Mol 

Surv 

<81.8 -- 

81.8 -- 

Mass; SVL ↑plasma thyroxine;↓ corticosterone 

Reported no exposure related mortalities; mortality not reported in terms of NOAEC/LOAEC 

Invalid due concentrattested 

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 re

solution prsolutions, wcontrol surInvalid due choice and experimenlevel of un

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 repreparationwater qualInvalid due and lack oexperimenlevel of un

Lenkowski et al2008 / 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 reanalysis of parametersInvalid due reporting oparameters 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 variable reone replicafor sex ratireported foreported Qualitative

higher loadand an occcoupled wtreatment gmetamorph
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

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 

Temperatupathologie(not controatrazine trerenewal; Utreatment alimited waQualitativeappears thatemperaturhave contras limited renewal pe

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 controf intersex controls apInvalid basgonadal inmortality 

Morgan 1996 / E63246 

X. laevis / Xenopus I (adults) / E for 96 hr 

F / 

40.8 

NA / 

(Y/NA); 

NS21

plastic 

NS (20 

E/petri 

dish) ; 24 

hr 

Metam Surv 

-- 11000 

or 1100 (4488 or 448 ai) 

LC50 = 100000 or 126000 (40800 or51408 ai) EC50 (abnormalities) = 33000 or <8000 ( 13464 or 3264 ai) 

Limited wvolume nosolubility; unknown Qualitative on limited 

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 coDMSO whcontained solvent); aatrazine praround colreported; utest termingrowth anaQualtiativehigher than 

21 Test concentrations measured but do not report results for control; unsure if analyzed 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

using a nonlimited reploading rat

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 caused or fdifferences DO ok, nitQualitativehigher than using a nonuncertainty 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 (Rohr et al 

were expoand test suany additio(water con(E81748) fQualitativeidentified uwhich the 

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 ecercariae eenvironmeresults, the control sugthe controlcontrol moQualtiativemortality nreported w(tadpoles ethan recomrelevancy 

Rohr et al. 2009 / E117315 

B. americanus / Field / LV 

T/ 

99 

ACTN 

(0.0002) / NS 6 TP/0.5L (96hr) Behav Surv 196 (M) -- >196 -- Fear queues (predator and parasite); activity No negativ

solvent bas

22 Solvent concentration was calculated by reviewer 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

(G25-27) for 4 d 

(N/Y); NS then 

1/3.5L; 

static 

Reported no effect on mortality with nearly 100% survival 

quality repQualitativeno negativ

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 (Rohr et al 

were expoand test suany additiocarryover e

for commeQualitativeidentified uwhich the 

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 survcontrol for embryo stuorganismsInvalid bassolvent consolvent in group; also higher than renewal pe

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 determhistology oand length 

the individnot the tanpooled resuQualitative

seemed wiammonia awith a 2X/organisms exposure h

Storrs and Pseudacris F / NA / PE 1 TP/ 0.1 Surv NDR 2.84 Survival- not dose responsive, for all Survival n23 Solvent concentration was calculated by reviewer 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

Kiesecker 2004 / E78290 

crucifer, R. sylvatica, B. americanus, R. clamitans / Field / TP (early stage G25-27) & (late stage G29-36) for ca. 30 d 

85 (Y/NA); 

NS 

or 0.5L; 

every 3 

days 

but wood frog, report sign. effects at 2.84, but not always at 25 or 64 

metamorpheliminated reported Qualitativesurvival rewater qualrate. 

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 westrogenic UncertaintQualitativelimited wathan recom72hr as wesolvent con

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 reuncertainty solvent; unthan nominQualitativelimited rep

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 

Storrs & Sdose respoUncertaintQualitativecomments 

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 

Changing the numbeQualitativechanging enumber of 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect with 320 μg/L atrazine than 0 μg/L nitrate 

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 docuendpoints, of the meathe numbeanimals noQualitativeneed for an

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 dmeasuremendpoint to fitness; useexperimenQualitativeneed for an

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 repvariability issues Invalid for Qualitativeremaining in endpoin

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 coprovided, cQualitative on lack of concentrat

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 wvessel not Qualitativelimited waperiod of 7recommen

Zaya et al. 2011a / E153847 

X.laevis / NS (Nasco?) / TP (NF47)-NF 

T/ 

NS 

NA / 

(Y/NA); 

NS 

glass 4 TP/1L; 3X/wk Metam Gro Behav 200 (N) 25 400 400 200 >400 Time to metamorphosis Mass; fat bodies Feeding Potential e

staging dufor feeding 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

58or 62 Sexdev 

Imm/inf Surv 

400 200 400 

>400 400 >400 

Sex ratio (gross morphology) ↑ Activated caspase 3 immuno(+) cells Survival 

liver size eQualitativelimited warecommenhours 

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 (actylCoA ehydrogenase and glucocorticoid receptor) Used organisms from Zaya et al

2011a (E153847) 

etiology of in this articsampling; Qualitativepotential breported wand a renew

Cosms Boone & BridgesBritton 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 poweQualitative 

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 dasome endpmeasuremQualitative 

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 i

likely; poteunknown 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 cohigher variQualitative 

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 coand length Qualitative 

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 

Continuaticoncerns; udifferent ti
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

higher occInvalid bas

Jooste et al 2005 / MRID 46965603 

X.laevis / Field (Adults) / TP -M

T/ 

MeOH 

(0.025%) / 

(N/Y); 

NS24

PE 800 TP / 1100L 

Metam Sexdev Surv 

30.8 (M) 30.8 NS 

>30.8 >30.8 NS 

Time to metamorphosis Testicular oocytes 

PreliminarUnusually temperaturHigher incInvalid bas

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 coarea where biochemicmetamorphexposed, nQualitative 

Relyea 2009 / E114296 

H.versicolor & R. pipiens / NS (collected as eggs) / TP-M 

T/ 

99 

EtOH 

(0.003) / 

(Y/Y); 

NS25

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 cthroughout measured vnot analyzpesticides much ethatanks. Qualitative 

Rohr and Crumine 2005 / E117319 

R.sylvatica/ Field / TP-M F / 

ACTN 

(0.00246) 

/ (Y/Y); 

NS26

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 cacclimatioQualitative 

Rohr et al. 2008a / E112910 

R.pipiens & R. clamitans / Field/ TP for 4 wks 

T/ 

98 

NS (Y/Y); 

NS27 plastic 

65 

amphibs/8

00L 

Imm/Inf Surv 

<117 117 

117 117 

↓ eosinophils ↑ trematode cycts (R. 

clam); ↓Melanomacrophage ↓ % survival for R. pipiens and no 

Unknown concentratUnknown initiation . 

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 26 Atrazine measured in treatment tanks; unclear if analyzed in control 27 Atrazine measured in treatment tanks; unclear if analyzed in con27trol 
Citation / 

ECOTOX(E)/ 

MRID 

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2

/ % 

ai 

Solvent 

(%)3 / 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results 7 IndentEndpoint NOEC 

(μg/L) 

LOEC (μg/L) 

Effect 

&<117 &>117 effect for R. clamitans

Qualitative 
22

2. Summary of Amphibian Open Literature as of the 2007 SAP on Amphibians 

Several of the open literature studies (e.g., Gibble et al. 201128; Koprivnikar 201029; 

Houck and Sessions 200630) examined potential effects of atrazine on varying parameters 

associated with immune response for aquatic-phase amphibians (African clawed frogs 

[Xenopus laevis] and Northern leopard frogs [Rana pipiens]). These studies also 

provided a limited amount of information on the effects of atrazine on test animal growth, 

development and survival. While the Gibble et al. 2011 study did not result in any 

adverse effects on measurement parameters at atrazine concentrations as high as 1000 μg 

a.i./L, Koprivniker 2010 reports interactive effects of parasitism and predation cues on 

growth and development at atrazine concentrations of 3 μg a.i./L. Additionally, leopard 

frog larvae exposed to 300 μg a.i./L exhibited mortality ranging between 95 – 100%. 

This study though is confounded by poor control survival, the use of plastic exposure 

systems and inconsistent test results. A study by Houck and Sessions 2006 examining 

the effect of atrazine exposure (1 and 10 μg a.i./L) for 4 or 8 weeks on the immune 

response R. pipiens using a hemolytic plaque assay and lymphocyte counts and reported 

effects at ≤1 μg a.i./L plaques (representing activated B-cells). Uncertainties in this study 

include information on the source and number of test animals; husbandry conditions 

were a major source of uncertainty as animals did not appear to feed during the study 

period. In Haridon et al. 199331 no effect on the number of micronucleated red blood 

cells was observed at atrazine concentration of 300μg/L; this study had limited water 

quality reported, appeared to use pseudoreplicates for statistical analysis and used 

dimethly sulfoxide as a solvent and it was not clear if the study included a solvent 

control. 

In another study by Koprivnikar et al. 200732, Rana sylvatica tadpoles were exposed to 

atrazine and Echinostoma trivolvis cercariae that were either also exposed to atrazine 

concentration or not (control cercariae). Additionally, the study authors state that no 

effect was observed on the prevelance of infection between atrazine groups and control 

cercariae. There was a significant difference in cyst abundance and intensity between 

control and 30 μg/L group exposed to control cercariae. The authors indicate that the 

presence of control cercariae affected tadpole activity whereas atrazine concentration did 

not affect tadpole activity as well as no interaction was found between effects of cercariae 

and atrazine. However, there was only an effect when the tadpoles but not the cercariae 

were exposed to atrazine, therefore, there is uncertainty in the environmental relevance of 

the study. In addition, based on the study design, a control group that was not exposed to 

 

28 Gibble, R.E. and K.N. Baer. 2011. Effects of atrazine, agricultural runoff, and selected effluents on antimicrobial 

activity of skin peptides in Xenopus laevis. Ecotox. Environ. Saf. 74: 593-599. (ECOTOX Ref No. 153856) 29 Koprivnikar, J. 2010. Interaction of environmental stressors impact survival and development of parasitized larval 

amphibians. Ecol. Appl. 20 (8), p 2263-2272. (ECOTOX Ref No. 153870) 30 Houck, A., and S.K. Sessions. 2006. Could atrazine affect the immune system of the frog, Rana pipiens? Bios. 77 

(4) 107-112 

31 Haridon J.L., M. Fernandez, V. Ferrier, and J. Bellan. 1993. Evaluation of the genotoxicity of Nnitrosoatrazine, N-nitrosodiethanolamine and their precursors in vivo using the newt micronucleus test. 

Wat. Res. 27(5). 855-862 

32 Koprivnikar, J.,  M. R. Forbes, and R. L. Baker. 2007. Contaminant effects on host‐parasite interactions:  

atrazine, frogs, and trematodes.  Environ. Toxicol. Chem.  26 (10):  2166‐2170
23

both atrazine and cercariae was tested whereas the control group exposed to cercariae had 

a mortality rate of 38%. 

In a study by Kerby and Storfer 200933, researchers examined the effects of atrazine (10, 

20 or 200 μg a.i./L) on the susceptibility of 4-wk old tiger salamander (Ambystoma 

tigrinum) larvae to a viral pathogen Ambystoma tigrinum virus (ATV) during a 3-wk 

exposure period. According to the study authors, significant interactive effects of 

atrazine by virus (p = 0.03) with atrazine slightly reducing mortality in no-virus 

treatments and slightly increasing mortality in virus treatments. The effects that were 

noted by the researchers did not appear to be dose responsive. Atrazine had no 

significant effect on infection rate nor did it significantly affect viral load. The study did 

not employ a solvent control although methanol was used as a co-solvent; additionally, 

the study was conducted in plastic containers. 

Rohr et al. 200834 examined the effects of static 4-wk exposure to atrazine at 102 μg/L on 

amphibians in 1,100-L plastic cattle tanks. Just before dosing, each test tank was 

prepared with four larval amphibians (5 Ambystoma maculatum, 20 Hyla versicolour, 20 

R. palustris and 20 R. clamitans) along with a limited number of other invertebrates. In 

order to simulate exposure to trematodes, each tank also received a caged planorbid snail 

shedding cercariae from the family Plagiorchidae; snails were rotated among tanks every 

other day for "homogenization" purposes although the snails were reported as not having 

shedded cercariae uniformly/consistently throughout the study period. The study also 

reports on a survey of eighteen wetlands in the Broadleaf Forest Ecoregion of Minnesota 

to examine 240 "plausible predictors" of the abundance of larval trematodes in northern 

leopard frogs (R. pipiens). In 1999, the authors characterized the biological diversity of 

the study sites and obtained water, sediment and recently metamorphosed frog tissue 

samples for parasite quantification and pathology. Frogs (Gosner Stage 46) were also 

examined for melanomacrophages and eosinophils. Results from the mesocosm studies 

indicated that atrazine significantly reduced phytoplankton chlorophyll a concentrations 

while periphyton chlorophyll a concentrations were significantly increased. Snail 

populations in the atrazine treated tanks were also significantly higher (4x) than in 

controls. According the study authors, this reduction in phytoplankton increased nutrient 

availability, water clarity and light penetration for periphyton. For R. palustris, 

specimens from the atrazine-treated mesocosms showed much lower liver 

melanomacrophages and eosinophil counts than specimens from the control tanks. 

Survival of R. palustris was also much lower in the atrazine-treated tanks (47.5% survival 

in the atrazine treatment group versus 70% survival in the controls). For R. clamitans, 

the study authors did not observe significant differences between the atrazine-treated 

tanks and the control tanks regarding liver melanomacrophages or elevated mortality; 

however, atrazine-treated mesocosms showed significant reductions in liver eosinophil 

counts. R. clamitans in atrazine treated tanks had significantly more plagiorchid 

 

33 Kerby, J.L and A. Storfer. 2009. Combined effects of atrazine and chlorpyrifos on susceptibility of the tiger 

salamander to Ambystoma tigrinum virus. Eco. Heal. (ECOTOX Ref No. 121497) 34 Rohr, J. R., A. M. Schotthoefer, T. R. Raffel, H. J. Carrick, N. Halstead, J. T. Hoverman, C. M. Johnson, L. B. Johnson, C.

Lieske, M. D. Piwoni, P. K. Schoff, and V. R. Beasley. 2008.  Agrochemicals increase trematode infections in a declining

amphibian species. Nature.  455(7217): 1235‐1239. (MRID 482611-31)
24

trematodes than in control tanks when controlling for the relationship between 

plagiorchid loads and eosinophils. Plagiorchid trematode infections were not reported for 

R. palustris. Based on the results of the wetland survey, the authors state that atrazine 

combined with desethylatrazine (DEA) was the best predictor of total larval trematode 

abundance in R. pipiens out of more than 240 measured "plausible predictors" of 

amphibian trematode infections. Additionally, atrazine combined with desethylatrazine 

was a significant negative predictor of melanomacrophages aggregate scores. The 

authors concluded that atrazine (plus DEA) can be a primary driver of larval trematode 

infections at concentrations commonly occurring in freshwater ecosystems (they also 

suggested that phosphate played a complementary role). The study authors proposed that 

atrazine increased exposure and susceptibility to larval trematodes by increasing the 

amounts of snail intermediate hosts (via increasing the periphyton) and by suppressing 

amphibian immune responses. Environmental conditions of the mesocosm studies 

discussed in the article are not well described and whether the experimental units were 

roughly equivalent prior to study initiation. 

A study tested the interaction of emerging infectious diseases and atrazine in long-toed 

salamanders (Ambystoma macrodactylum) (Forson and Storfer 200635). The effects of 

atrazine and virus was tested on mass and snout-vent length (SVL) at metamorphosis and 

larval period as well as on rates of mortality and viral infectivity. Larvae exposed to both 

atrazine and ATV had lower levels of mortality and ATV infectivity (13.3% across all 3 

atrazine concentrations) compared to larvae exposed to virus alone (25%), suggesting 

atrazine may compromise virus efficacy. The highest atrazine level (184 μg/L), but not 

at 18.4 μg/L accelerated metamorphosis and reduced mass and SVL at metamorphosis 

relative to controls. Exposure to ATV also significantly reduced SVL at metamorphosis. 

Atrazine alone had no significant effect on mortality. In this study there was limited 

reported water quality and the loading rate was higher than recommended (1 tadpole/L). 

In another study aby Forson and Storfer 200636 Ambystoma tigrinum larvae were 

exposed to atrazine concentrations, one sodium nitrate concentration and either none or 1 

x 104 plaque forming units/mL of A. tigrinum nebulosum virus (ATV). The percent 

infected larvae were significantly greater at 16 μg/L compared to the control (but not 1.6 

or 160 μg/L). The number of leukocytes was decreased at 16 and 160 μg/L compared to 

the control in the viral treated organisms. There were no significant differences on viral 

copy between any atrazine treatment group. For atrazine only exposures, there were no 

effects on mass, SVL or larval period. Limited water quality was reported and based on 

information provided, loading was assumed to be greater than recommended 

 

35 Forson, D. and Storfer, A. (2006). Effects of Atrazine and Iridovirus Infection on Survival and LifeHistory Traits of the Long-Toed Salamander (Ambystoma macrodactylum). Environ.Toxicol.Chem. 25: 

168-173 

36 Forson, D.D. and A. Storfer. 2006. Atrazine increases ranavirus susceptibility in the tiger salamander, 

Ambystoma tigrinum. Ecol. App. 16(6): 2325-2332. 
25

In Kiesecker 200237, wood frogs (Rana sylvatica) were exposed to atrazine and Ribeiroia 

sp or Telorchis sp cercariae and a study authors indicate atrazine resulted in a decrease in 

eosinophils and an increase in the proportion of cercariae that encysted was observed at 3 

and 30 μg/L. This controls in this study also contained the cercariae and an additional 

control that did not contain atrazine or cercariae to observed the performance of the 

organisms in the absence of stressors was not tested. Results on mortality were not 

reported and limited water quality was reported. 

Rana pipiens were exposed to atrazine and then treated with an inflammatory mediator to 

examine the activity of white blood cells which was used as an indicator of innate 

immune response (Brodkin et al. 2007)38. A decrease in the number of highly phagocytic 

cells was significant less at all treatment groups, 0.01-10 μg/L, compared to the 

thioglycollate stimulated control. In addition, at 21 μg/L, only treatment group, atrazine 

significantly diminished innate immune response when compared to frogs not exposed to 

atrazine but injected with thioglycollate (an inflammatory mediator) as the number of 

peritoneal exudates cells was significantly decreased. A treatment group that did not 

contain atrazine or was not treated further was not used in this study. While the study 

reported a change in white blood cells, these endpoints were not clearly and quantiatively 

linked to effects on survival, growth or reproduction in this study. 

Amphibian development was examined in several of the studies. In a study by 

Lenkowski and McLaughlin 201039, the study authors report that acute (6 – 48 hr) 

exposure of X. laevis larvae to 35,000 μg a.i./L affected development (structural integrity 

of cartilage and muscle). While atrazine did not inhibit development, the study authors 

assert that atrazine exposure results in an alteration of differentiated tissues (cranial and 

muscle) and they hypothesize that this disruption may be through modulation of matrix 

metalloproteinases. The atrazine concentrations tested in this study are high and the 

study relied on dimethylsulfoxide (DMSO). In another study by Lenkowski et al. 201040, 

X. laevis NF Stage 40 larvae were exposed for 48 hrs to atrazine concentrations of 350, 

3500, 10000 and 35000 μg a.i./L. According to the study authors, intestinal 

malformations and edemas were significantly increased in the three highest test 

concentrations and cranial muscles were reduced in the highest test concentration 

compared to controls. Although the authors speculate that muscle degradation may be 

atrazine on altered cell signaling, no data are provided to support this assertion. Similar 

to the previous study by Lenkowski, the various treatment solutions contained DMSO 

and treatment concentrations at which effects are noted are relatively high; additionally, 

environmental conditions used in the study are not well characterized. 

 

37 Kiesecker J.M. 2002. Synergism between trematode infection and pesticide exposure: A link to 

amphibian limb deformities. Proc Nat. Acad. Sci. 99 (15): 9900-9944 38 Brodkin, M.A., H. Madhoun, M. Rameswaran, and I. Vatnick. 2007. Atrazine is an immune disruptor in 

adult northern leopard frogs (Rana pipiens). Environ. Toxicol. Chem. 26 (1):80-84 

39 Lenkowski, J.R. and K. A. McLaughlin. 2010. Acute atrazine exposure disrupts matrix metalloproteinases and 

retinoin signaling during organ morphogenesis in Xenopus laevis. J. App. Toxicol. 30: 582-589 (ECOTOX Ref No. 

153851) 

40 Lenkowski, J.R, G.Sanchez-Bravo, K.A. McLaughlin. 2010. Low concentration of atrazine, glyphosate, 2,4-

dischlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis. 

J. Environ. Sci. 22(9): 1305-1308. ECOTOX Ref No. 153876) 
26

In a study by Kloas et al. 2009, 4-day exposure of X. laevis tadpoles to atrazine at 25 μg 

a.i./L had no significant effect on aromatase gene expression in the brain or kidneygonadal complex of undifferentiated (NF Stage 48) and differentiated (NF Stage 58) 

larvae. 

Williams and Semlitsch 201041 examined the effects of atrazine exposure throughout the 

larval period to survival differences or life history trait alterations in three species: 

American toads (Bufo americanus), western chorus frog (Pseudacris triseriata) and gray 

treefrogs (Hyla versicolor). Larvae were exposed from Gosner Stage 25 to 

metamorphosis (Gosner Stage 42) to formulated atrazine at mean measured 

concentrations of 0.28 and 1.25 μg a.i./L. Chronic exposure to atrazine induced no 

significant effects on survival, mass a metamorphosis or larval period length at the 

concentrations tested. 

Storrs-Mendez and Semlitsch 200942 examined gonadal morphology of American toads 

(Bufo americanus), gray treefrogs (Hyla versicolor) and Southern leopard frogs (Rana 

sphenocephala) treated with atrazine (1, 3 or 30 μg a.i./L) or estradiol to determine 

whether amphibians with an accelerated rate of sexual differentiation were more 

susceptible to atrazine as larvae and metamorphs than species with slower rates of sexual 

differentiation. Exposures were initiated at Gosner stage 25 and animals were sampled at 

3-wk intervals until the completion of metamorphosis. For R. sphenocephala (long larval 

period with accelerated rate of gonadal development, animals were sampled at 3, 6, 9 wks 

and at the completion of metamorphosis; for B. americanus, which had the shortest larval 

period rate of differentiation, and H. versicolor, which have a intermediate larval period 

and slow rate of differentiation, samples were collected at 3 wks and at the completion of 

metamorphosis. The data indicate that for B. americanus the percentage of females at 

metamorphsis was higher in toads treated with atrazine at 30 μg a.i./L compared to 

controls; for H. versicolor, the percentage of females at metamorphosis was higher in 

treefrogs treated with 1 and 30 μg a.i./L, but not at 3 μg a.i./L. For R. sphenocephala, 

there were no significant differences in sex ratio at 6 wks while at 9 wks there were 

significant differences in the sex ratio of frogs treated with atrazine at 3 and 30 μg a.i./L, 

but not at 1 μg a.i./L. The percentage of Rana exhibiting testicular oocytes and ovotestes 

varied across control, estradiol and atrazine-treated animals; however, the highest 

percentages of these effects were observed at 9 weeks. By metamorphosis though there 

were no significant differences in sex ratio between atrazine-treated and control Rana; 

however, ovotestes were observed in both controls and the 30 μg a.i./L atrazine treated 

animals. In juvenile frogs, only B. americanus treated with atrazine at 1 μg a.i./L 

exhibited significantly different sex ratio (higher percentage of females) relative to 

controls. The authors concluded that a phase of intersex gonads is normal during leopard 

frog (rapid differentiation) sexual development and that intersex gonads were found in 

juvenile stages of toads and treefrogs representing animals with slow and basic rates of 

gonadal differentiation. Treefrogs exhibited underdeveloped testes in a limited number 

 

41 Williams, B. K. and R. D. Semlitsch. 2010. Larval Responses of Three Midwestern Anurans to Chronic, Low-Dose 

Exposures of Four Herbicides. Arch. Environ. Contam. Toxicol. 58: 819 - 827 (ECOTOX Ref. No. 153825) 42 Storrs-Méndez, S. I. and R. D. Semlitsch. 2009. Intersex Gonads in Frogs: Understanding the Time Course of 

Natural Development and Role of Endocrine Disruptors. Journal of Experimental Zoology (Mol Dev Evol) 312B: 1 -

11. (ECOTOX Ref. No. 153841) 
27

of animals exposed to 1 μg a.i./L atrazine (one animal) or estradiol (2 animals) while 

Rana had underdeveloped testes in juveniles exposed to atrazine at 1 μg a.i./L and 10 μg 

a.i./L. The absence of a negative control and the use of plastic exposure containers in this 

study are considered major limitations in the study and the methods section does not 

describe how juvenile exposures were conducted. Measured concentrations of atrazine in 

the study with Bufo larvae ranged from 2.5 - 4.2 times the nominal test concentrations. 

For juvenile studies, measured concentrations ranges from 1.5 - 4 times higher than 

nominal. Depending on the species and sampling period, control animals appeared to 

reflect relatively skewed sex ratios. The fluctuating sex ratios in ethanol controls suggest 

that gonadal development was inconclusive prior to metamorphosis and that it may not 

serve as a reliable indicator of development in larvae. Although the authors indicate that 

underdeveloped testes are unusual and allude to an increased incidence in atrazine-treated 

animals, the number of affected animals is relatively low and it is uncertain whether 

incidence of underdeveloped testes is a reliable measurement endpoint. 

In a study by Zaya et al. 2011a

43, the effects of atrazine on survival, growth and 

development of X. laevis were examined. A total of six studies were conducted where 

NF Stage 47 larvae were exposed to atrazine concentrations ranging from 25 to 400 μg 

a.i./L. Depending on the experiment, larvae were sacrificed at either NF Stage 58 or 62 

at which time animals were measured for snout-vent-length (SVL) weighed and their age 

(in days) recorded. Gender was determined by gross morphology and the liver and fat 

bodies were evaluated with differential staining for glycogen and caspase-3. There were 

no significant differences in body weights in larvae exposed to 25 and 200 μg a.i./L 

relative to controls; whereas animals exposed to atrazine at 400 μg a.i./L exhibited 

significantly lower body weights beginning at 3 weeks of exposure with some animals 

exhibiting lower body weights as early as 72 hrs of exposure. There were no significant 

differences in SVL in animals treated with 25 and 200 μg a.i./L. Although body weights 

were consistently lower than control for animals exposed to 400 μg a.i./L only one of the 

five studies exhibited significantly reduced SVL relative to controls. There were no 

differences in metamorphic stages between controls and animals treated with 25 and 200 

μg a.i./L; changes in weekly metamorphic stage were inconsistently noted at 400 μg 

a.i./L. There were no differences in sex ratio and no intersex animals noted between 

control and atrazine-treated animals. For both fat bodies and livers, there were no 

significant differences between controls and larvae treated with 25 μg a.i./L. However, 

for larvae exposed to 200 and 400 μg a.i./L exhibited significantly lower fat body weights 

relative to controls. In one of two studies liver weighs were significantly lower than 

controls for larvae treated with 200 μg a.i./L, and in two of four studies liver weights 

were significantly lower than controls for larvae treated with 400 μg a.i./L. No 

pathological changes were noted in the fat body and liver histology of atrazine-treated 

larvae and there were no differences in glycogen or lipid content of the livers; however, 

the authors noted that in one study, larvae exposed to 400 μg a.i./L had significantly more 

caspase-3 activated immunopositive cells relative to controls. The authors concluded that 

based on reduced liver and fat body sizes, the NOEC for the study is 25 μg a.i./L. 

 

43 Zaya, R. M., Z. Amini, A. S. Whitaker, S. Kohler and C. F. Ide. 2011a. Atrazine exposure affects growth, body 

condition and liver health in Xenopus laevis tadpoles. Aquatic Toxicology 104: 243 – 253. (ECOTOX Ref. No. 

153847) 
28

Storrs et al. 200944 examined the effect of atrazine exposure through soil on the terrestrial 

phase of American toads (B. americanus). Wild-caught toads were exposed to soil 

treated with atrazine at 80 or 1430 μg/kg for two consecutive nights and their behavior 

was then monitored for 12 hours to determine whether the toads preferred treated or 

untreated soil. A subgroup of the toads (fasted for 72 hours) used in the soil study was 

placed in 1-L glass jars containing water with radio-labeled atrazine at 460 μg/L plus an 

dry area. The pelvic patch of the test animals was recorded and toads were taken for 

analysis at 2, 4, 8, 12 and 24 hrs after exposure. At 8 and 12 hours, any remaining water 

in the exposure jars was replaced with clean water; toad internal organs were then 

sampled to determine the distribution of labeled atrazine. In the soil preference study 

measured atrazine levels in the treated soil were 59% lower than nominal and there was 

no significant difference between the number of toad on atrazine-treated versus untreated 

soil, i.e., the toads did not exhibit a statistically significant preference for treated versus 

untreated soil. In the uptake and elimination study, the highest percentage of 

radioactivity was in the intestine. According to the study authors, the highest 

concentration was in the gall bladder and intestine at 8 hrs although figures in the 

publication suggest that peak levels occurred at 12 hours in both tissues; peak 

concentrations in other tissues (i.e., liver, stomach and kidney) occurred at 2 hours. This 

study is seriously confounded by the absence of a solvent (ethanol) control, the reuse of 

previously tested animals (albeit 4 months previous) and uncertainty regarding the nature 

of radio-labeled residues (i.e., parent atrazine or a particular transformation product). 

In Rohr et al. 200945, tadpoles were evaluated to determine if exposed to atrazine for 4 

days altered their perception (responses to) of risk of predation and parasitism. Previous 

exposure to atrazine did not significantly affect responses for any of the cues. Cercariae 

alone and infected snail treatments significantly increased tadpole activity (by number of 

segments crossed); but other treatments were not significantly different including 

previous exposure to atrazine. The study provides information on the survival of 

tadpoles to atrazine for 4 days, however, the results of the behavioral studies cannot be 

used for the risk assessment endpoints of survival, growth or reproduction as direct 

measures of these endpoints were not conducted (exposures to queues were for one hour 

only). 

In mesocosm studies (Langlois et al. 201046) with wild-caught R. pipiens, larvae (Gosner 

Stage [GS] 25) were exposed through metamorphosis to nominal atrazine concentrations 

of 0. 0.1 and 1.8 μg a.i./L in addition to control and 17-β estradiol (EE2). According to 

the study authors, survival was significantly lower for larvae exposed to 1.8 μg a.i./L 

(66%) and EE2 (65%) compared to controls (76 – 79%) with significantly fewer larvae 

reaching metamorphosis in both the atrazine-exposed (45 – 50%) groups as well as the 

 

44 Storrs Méndez, S.I., D.E. Tillitt, T.A.G Rittenhouse, R.D. Semlitsch. 2009. Behavioral response and kinetics of 

terrestrial atrazine exposure in American toads (Bufo americanus). Arch. Environ. Contam. Toxicol. 57: 590-

597(ECOTOX Ref No. 118898).

45 Rohr, J. R., A. Swan, T.R. Raffel, and P.J. Hudson. 2009. Parasites, info-disruption, and the ecology of 

fear. Ocecologia. 159: 447-454 46 Langlois, V.S., A.C. Carew, B.D. Pauli, M.G. Wade, G.M. Cooke, V.L. Trudeau. 2010. Low levels of the herbicide 

atrazine alter sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms. 

Eviron. Heal. Perspec. 118 (4): 552-557. (ECOTOX Ref No. 120046)
29

EE2 treatment (55%) compared to controls (76 – 85%). The study authors also noted that 

the sex ratio for the 1.8 μg a.i./L (male:female; 1:1.14) was significantly different (higher 

percentage of females) compared to controls (1:0.6). The sex ratio results are difficult to 

understand though since the EE2-treated group (1:0.9) did not differ substantially from 

controls. Also, although the authors report 22% of the males in the EE2 group had 

testicular oocytes, no testicular oocytes were observed in the other treatments; this 

observation does not appear to be consistent with the increased number of females 

reported in the 1.8 μg a.i./L atrazine treatment. Atrazine did not affect aromatase 

(cyp19mRNA) expression in pre-metamorphic larval (G34 and G45) brain tissue; 

expression of estrogen receptor alpha (eralpha) in the brains but not livers of G34 larvae 

was higher (2.5x) than controls in larvae treated with 1.8 μg a.i./L but not in brains or 

livers from G45 larvae. Although the authors assert that the study provides evidence that 

atrazine may feminize R. pipiens exposed to low levels of atrazine and that this effect is 

associated with changes in brain eralpha mRNA and liver steroid 5-beta reductase 

(srd5beta) activity, EE2-treated animals did not appear to exhibit significantly different 

levels the expression of these genes even though the sex ratio indicated feminization. 

Insufficient data are provided in the study to support the study author’s hypothesis 

regarding the potential role of eralpha and srd5beta in altered sex ratios/feminization. 

Also, although the authors assert that they present information that atrazine alters the 

thyroid axis by affecting metamorphic success and that thyroid hormone-related 

deiodinase gene expression (dio3 mRNA) in G34 tadpole tails, the reduced metamorphic 

rate at 0.1 μg a.i./L was higher than that observed at 1.8 μg a.i./L yet dio3 mRNA was not 

significantly reduced at 0.1 μg a.i./L. The study is confounded by atrazine contamination 

of the controls and differential (4o

C) water temperatures between the control and 1.8 μg 

a.i./L atrazine treatment. Also, there appeared to be a differential survival between males 

and females in the controls. 

Mesocosms (11.3-L capacity polyethylene tubs) were used by Rohr and Crumrine 200547

to determine whether a 4-wk static exposure to atrazine at 25 μg/L affects the pond 

community when the presence of wild-caught wood frog tadpoles (Rana sylvatica; 

Gosner Stage 23 - 26), adult snails (Planorbella trivolvis) are dragonfly larvae (Anax 

junius; caged) are varied. The study reports that the density of community members was 

at the high end of regional densities. Although the study employed an solvent (acetone) 

control, it did not employ a negative control. Mesocosms were retreated with atrazine 

(50 μg/L) after the initial 2 weeks. Atrazine concentrations were not monitored during 

the study and the study authors report that nominal concentration were likely higher due 

to heavy rainfall that occurred during the study. Periphytic algae were significantly 

reduced (roughly 15% decrease in chlorophyll a) and water clarity was greater in 

atrazine-treated mesocosms. Lower densitities (roughly a 24% decrease) of dipteran 

larvae (Polypedilum) were observed in atrazine-treated tanks while atrazine had no 

significant effect on the abundance of Daphnia spp. and other invertebrates including 

abundance and longevity of dragonfly larvae. The final mass of snails was significantly 

 

47 Rohr, J. R. and P. W. Crumrine. 2005. Effects of an herbicide and an insecticide on pond community structure and

processes. Ecological Applications. 15(4): 1135‐1147. (MRID 48261130)
30

reduced in atrazine treated tanks and when adjusted for mass, fewer egg masses per 

surviving snail were deposited in atrazine-treated mesocosms. Other than significantly 

fewer (roughly 6% difference) tadpoles were hiding in atrazine-treated mesocoms 

compared to the solvent control, there were no other significant effects of atrazine on 

tadpole activity. Although the authors report that wood frog development was slowed by 

atrazine, the difference between Gosner stage of atrazine-treated and solvent control frogs 

was not statistically significant. Additionally, atrazine treatment did not have a 

significant effect on final tadpole mass. According to the study authors, the direct effects 

of atrazine in reducing periphyton (as measured by chlorphyll a) resulted in direct effects 

on snails, tadpoles and dipteran (Polypedilum) larvae. The prior exposure history of the 

organisms used in the study is uncertain and the water used in the study was not analyzed 

for other potential contaminants combined with the absence of a negative control are 

potential limitations in the study. 

For Boone and James 200348 Atrazine exposure at 200 μg/L, the only concentration 

tested, did not influence the mass, SVL, developmental stage, or survival of spotted 

salamanders(Ambystoma maculatum) in outdoor a mesocosm study. For leopard frogs

Rana sphenocephala, atrazine exposure did not significantly influence survival. 

However, survival was significantly influenced by an atrazine by population density 

interaction where atrazine exposure decreased survival in the high-density ponds but not 

the low-density ponds relative to the control ponds (treatments without atrazine) at 200 

μg/L. Mass significantly decreased with atrazine exposure compared to the controls. 

Results of the competitive controls indicated that no difference occurred between spotted 

salamanders reared alone or with southern leopard frogs. 

 In the Relyea 200949 mesocosm study, for leopard frog tadpoles (R. pipiens), there was 

not a significant difference between the atrazine treatment and the no-pesticide treatments 

(i.e. negative and ethanol control tanks) regarding survival to metamorphosis, mass at 

metamorphosis, and time to metamorphosis. For gray tree frog tadpoles (H. versicolor), 

there was not a significant difference between the atrazine treatment and the no-pesticide 

treatments regarding survival to metamorphosis and time to metamorphosis. However, 

for mass at metamorphosis, gray tree frogs were larger in tanks treated with atrazine 

compared to those in the negative and ethanol control tanks at 10 μg/L. 

In a study by Brodeur et al. 200950, South American toads (Rhinella arenarum) at 

different levels of development (Gosner Stages 4, 25 and 38 - 39) were exposed to 

atrazine concentrations ranging between 0.1 – 20,000 μg a.i./L. Exposures with Stage 4 

(embryos) toads were conducted for 21 days. The study with larvae at Gosner Stage 38 

– 39 was conducted to completion of metamorphosis. Exposure from Stage 4 resulted in 

 

48 Boone, M. D. and S. M. James. 2003. Interactions of an insecticide, herbicide, and natural stressors in 

amphibian community mesocosms. Ecological Applications. 13(3): 829-841. (MRID 48261104)

49 Relyea, R. A. 2009. A cocktail of contaminants: how mixtures of pesticides at low concentrations affect 

aquatic communities. Oecologia 159(2): 363-376. 

50Brodeur, J.C., G. Svartz, C.S. Perez-Coll, D.J.G. Marino, J. Herkovits 2009. Comparative susceptibility to atrazine of 

three developmental stages of Rhinella arenarum and influence on metamorphosis: Non-monotonous acceleration of 

the time to climax and delayed tail resorption. Aquatic Toxicology. 91 (161-170). (ECOTOX Ref. No. 112588)
31

10-, 14- and 21-day LC50 values of 25370, 14410 and 7150 μg a.i./L, respectively. 

Exposure of Stage 25 larvae resulted in 4-, 7-, 10-, 14- and 21-day LC50 values of 27160, 

20110, 14480, 7030 and 2320 μg a.i./L, respectively. Exposure of Stage 38 – 39 larvae 

resulted in 14-, 21-, and 28-day LC50 values of 18270, 14430 and 12630 μg a.i./L, 

respectively. According to the study authors, Stage 4 embryos, 10 days of exposure were 

needed to observe significant decreases in survival whereas Stage 25 larvae exhibited 

significant decreases in survival after 4 days of exposure. Of the three stages tested, 

Stage 25 were reported to be the most sensitive. None of the larvae in the 15,000 and 

20000 μg a.i./L reached metamorphosis and only 20% of the larvae in the 10000 μg a.i./L 

reached metamorphosis. According to the study authors, the time to reach Stage 42 was 

accelerated by atrazine with a U-shaped curve with 100 and 1000 μg a.i./L having faster 

times and the 5000 μg a.i./L having no difference compared to controls. The authors 

state that larvae exposed to 1000 and 5000 μg a.i./L exhibit a delay in metamorphosis of 

4 to 6 days in order for 80 – 90% of the animals to complete metamorphosis. Based on 

their data, the authors conclude that atrazine is unlikely to cause direct mortality to R. 

arenarum under typical use conditions. Stage 4 embryos required de-jellying of the eggs 

and it is uncertain how this process affects exposure to the test substance. Also, although 

the time required for 50% of the larvae to reach Stage 42 and/or complete metamorphosis 

was significantly different from controls for one or more of the atrazine treatment groups 

not affected by survival, there was no significant difference between these groups for the 

duration between when 50% of the test animals reached Stage 42 and the completion of 

metamorphosis. 

Hyla versicolor tadpoles (Gosner stage 25) were exposed to atrazine and exposure or not 

to a nonlethal predator, dragonfly larva (LaFiandra et al. 2008)51. The presence of the 

nonlethal predator significantly reduced the proportion of tadpoles that initiated 

metamorphosis at 200 μg/L, but not 20 μg/L. However, while loading was within 

recommended limits, limited water quality was reported with a relatively long renewal 

period (50% renewal of solution weekly). 

Xenopus laevis tadpoles were exposed to atrazine and tadpoles were staged weekly and 

upon complete metamorphosis, total length and time to metamorphosis was measured 

(Oka et al. 2008)52. Stage 66 frogs were sexed by visual observation of gross 

morphology; after visual observation, gonads were removed for histological analysis or 

biochemical analysis. . After metamorphosis, the sex ratio was significantly different for 

the 10 and 100 μg/L atrazine groups, but not the 1.0 μg/L group, compared to the 

control; no hermaphroditism was observed. According to the study authors, evaluation of 

sex by gross morphology was corroborated by histological analysis. Time to 

metamorphosis was similar among treatment groups and there did not appear to be an 

acceleration or inhibition of development. 

 

51 LaFiandra, E.M, K.J. Babbitt, and S.A. Sower. 2008. Effects of atrazine on anuran development are 

altered by the presence of a nonlethal predator. J. Toxicol. Environ. Health, Part A. 71: 505-511 

52 Oka, T., O. Tooi, N. Mitsui, M. Miyahara, Y. Ohnishi, M. Takase, A. Kashiwagi, T. Shinkai, N. Santo, 

T. Iguchi. 2008 Effect of atrzine on metamorphosis and sexual differentiation in Xenopus laevis. Aquat. 

Toxicol. 87: 215-226 
32

In Allran and Karasov 200053 Rana pipiens larvae were exposed to atrazine and nitrate 

from Gosner stage 25 till metamorphosis. According to the authors, atrazine did not 

affect percent metamorphosis, time to metamorphosis, percent survival, mass at 

metamorphosis or hematocrit at concentrations up to 200 μg/L; while there was no 

consistent effect of atrazine on growth, there was significant atrazine by time affect and 

an atrazine by nitrate by time effect however at what time points and concentrations were 

not reported. For the metamorphosis and mass endpoints, statistical analyses were 

conducted on 50% of the organisms at test termination and the reason for this is not clear. 

In another study by Allran and Karasov 200154 adult R. pipiens frogs had significantly 

higher buccal ventilation (breaths/minute) at 4,300 μg/L. Also, for R. pipiens, R. 

sylvatica and Bufo americanus) a significant increase in deformaties at 96 hour post hatch 

was observed at 4,300 μg/L, although there was no difference in hatchability or mortality 

at 96 hours post hatch. Raw data was not provided and low levels of atrazine was 

measured (up to 0.06 μg/L) at the frog collection site. 

In Larson et al. 199855, tiger salamanders larvae (Ambystoma tigrunum) exposed to 81.8 

μg/L atrazine reached stage 4 during development later than control but at a size and 

weight compared to control; larvae at 300 μg/L progressed at same rate as controls but 

were smaller and weighed less than controls. Plasma thyroxine was elevated in both 

atrazine-exposed groups compared to the control, and plasma corticosterone was 

depressed in the 81.8 μg/L treatment compared w/ both the control and the 300 μg/L 

treatment group. Atrazine was detected in the control sample at 0.23 ± 0.21 μg/L and no 

negative control was tested and the amount of solvent in the treatment groups is 

unknown. 

Sporlyarich et al. 201056 examined the effects of atrazine on growth, development and 

sex ratio of developing spotted marsh frogs (Limnodynastes tasmaniensis) from Gosner 

Stage 28 (early limb bud emergence) to Gosner Stage 42 (tail resorption). The authors 

reported that atrazine concentrations ranging from 0.1 to 30 μg a.i./L had no observable 

effect on the mean rate of development, sex ratio or growth (total body length) of the 

spotted marsh frog; however, testicular oocytes were observed in one male at Gosner 

Stage 44 after 4 wks of exposure to atrazine at 3 μg a.i./L. 

 

53 Allran, J.W., W.H. Karasov. 2000. Effects of atrazine and nitrate on northern leopard frog (Rana 

pipiens) larvase exposed in the laboratory from posthatch through metamorphosis. Environ. Tox. Chem. 

19(11): 2850-2855 

54 Allran J.W and W.H. Karasov. 2001. Effects of atrazine on embryos, larvae, and adults of anuran 

amphibians. Environ. Toxicol chem. 20(4): 769-775 

55 Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and Hamilton, S. J. (1998). Effects of the 

Herbicide Atrazine on Ambystoma tigrinum Metamorphosis: Duration, Larval Growth, and Hormonal 

Response. Physiol.Zool. 71: 671-679 56 Spolyarich, N., R. Hyne, S. Wilson, C. Palmer and M. Byrne. 2010. Growth, development and sex retios of Spotted 

Marsh Frog (Limnodynastes tasmaniensis) larvae exposed to atrazine and a herbicide mixture. Chemosphere 78: 807 - 

813. (ECOTOX Ref. No. 153638) 
33

A second paper by Zaya et al. 2011b57 examines the potential effects of atrazine on 

energy homeostatis in X. laevis by measuring gene expression from samples collected in 

a previous studies (Langerveld et al. 200958 and Zaya et al. 2011a) where NF Stage 47 

larvae were exposed to atrazine at 200 and 400 μg a.i./L. Some of the data collected in 

the study were based on relatively short exposure durations (24, 48, 72 hrs and 14 days). 

According to the study authors genes associated with synthesis and degradation of ketone 

bodies, the urea cycle, regulation of autophagy, citric acid cycle, glutathione metabolism 

and metabolism of a number of amino acids were upregulated while genes associated 

with glycolysis/glucogenesis, fatty acid elongation (mitochondirial and ribosomal) were 

down-regulated. The authors noted that based on their previous publication (Zaya et al. 

2011a), larvae were significantly smaller within 72 hrs of exposure and at NF Stage 62 

had depleted fat stores at the end of exposure. Of the six genes examined at 24, 48, 72 

hrs and 14 days of exposure in one study, the only significant difference was in acyl-CoA 

dehydrogenase and glucocorticoid receptor at 24 hrs. In studies examining ATP levels, 

there were no significant differences noted between control and atrazine-treated animals. 

The authors conclude that based on changes in gene expression, larvae exposed to 400 μg 

a.i./L experience an energy imbalance starting early in exposure; however, they are 

uncertain regarding the etiology of this energy imbalance. The article refers to 

Langerveld et al. 2009 to support claims that atrazine exposure at 400 μg/L resulted in 

significant reductions in survival and growth (weight, length and fat body size). 

However, the Langerveld et al. study has major uncertainties associated with it especially 

given that so many of the test animals were not surviving to metamorphosis that the 

protocol had to be adjusted to work with younger animals. Based on the N values in this 

study, it suggests that overall mortality of test animals in control and atrazine-treated 

groups was 63% and 57%, respectively. This further suggests that environmental 

conditions in the study were not sufficient to support animal survival. In studies where 

environmental conditions are poor and test animals are struggling to survive, it is 

uncertain whether any meaningful relationships can be evaluated for specific chemicals 

within that environment. As such, the Lagerveld et al. 2009 study would be classified as 

invalid. The Zaya article hypothesizes that the etiology of the "apparent energy 

imbalance" could be due to detoxification and excretion of atrazine and/or endocrine 

disruption is broad, and with respect to endocrine disruption, it is speculative. Given that 

the authors acknowledge that the glutathion [detoxification] pathway on which atrazine is 

subject to is energy demanding, it may be a more plausible explanation for the effects 

they report. It is uncertain whether the study results support the statement that "clearly, 

acutely and chronically exposed tadpoles are compromised, decreasing their ability to 

survive the stresses of metamorphosis and/or to adequately support reproduction. 

Although the authors state that atrazine-exposure resulted in depleted fat stores, the 

preceding publication by Zaya et al. 2011 did not indicate any significant difference in 

histological lipid staining; fat bodies in larvae exposed to atrazine at 400 μg/L were 

significantly [smaller]; however, it is unclear what that reduction in percent of body 

 

57 Zaya, R. M., Z. Amini, A. S. Whitaker, and C. F. Ide. 2011b Exposure to atrazine affects the expression of key 

genes in metabolic pathways integral to energy homeostasis in Xenopus laevis tadpoles. Aquatic Toxicology 104: 254 

– 262. (ECOTOX Ref. No. 153848) 

58 Langerveld, A.J., Celestine, R., Zaya, R., Mihalko, D., Ide, C.F., 2009. Chronic exposure to high levels of atrazine 

alters expression of genes that regulate immune an growth-related functions in developing Xenopus laevis tadpoles. 

Environ. Res.109, 379–389. 
34

weight was associated with. Additionally, the authors state that the rate of apoptosis was 

increased in NF Stage 62 larvae; however, the companion article by Zaya et al. 2011a

only speculated that increased caspase-3 activity would lead to increased apoptosis in 

spite of the fact that no pathology was observed through liver histology. 

Rohr et al. 201159 examined the potential interaction of temperature and atrazine 

exposure on growth, survival, hatching and metamorophic timing of the streamside 

salamander (Ambystoma barbouri). Salamanders were exposed from eggs (embryos) to 

atrazine concentrations of 4, 40 and 400 μg a.i./L at 13 or 19o

C for 49 days until all 

embryos hatched or were dead. In a separate experiment, larvae were exposed to the 

same concentrations of atrazine at 16, 19, 22 or 25oC. In the 25oC treatment, half of the 

larvae were exposed for 19 days while the remainder were exposed until completion of 

metamorphosis. According to the study authors, hatching and larval mass were 

significantly reduced in embryos exposed to 400 μg a.i./L relative to controls; larval 

survival was reduced for embryos exposed to 4, 40 and 400 μg a.i./L. Atrazine exposure 

however, did not significantly affect larval survival; although the authors indicated that 

no significant interaction was detected between temperature and timing of hatch, 

embryonic survival or larval survival, larval mass was significantly higher at 19o

C 

compared to 13oc and embryos hatched sooner at 19o

C. Based on the larval studies, the 

study authors conclude that atrazine exposure significantly delayed metamorphosis and 

reduced mass at metamorphosis and that while temperature was positively associated 

with developmental rate it did not significantly affect size at metamorphosis. Although 

the author assert that there was a trend for atrazine to be a significant negative predictor 

of larval survival, this study did not detect any statistically significant difference that 

could be used to support that assertion. The study data indicated that increasing 

temperature decreased the effect of atrazine on both timing of and size at metamophosis. 

The authors conclude that increasing atrazine concentration delayed hatching and 

metamorphosis, reduced growth and decreased embryonic and larval survival and that 

with the exception of growth, increasing temperature did not enhance the toxicity of 

atrazine, but did ameliorate adverse effects of atrazine by accelerating development and 

reducing the duration of atrazine exposure. The absence of a negative control and 

apparently poor solvent control survival represent major limitations in this study. 

Storrs and Kiesecker 200460 examined exposure (30 days) to four species of 

tadpolesfrogs including spring peepers (Pseudacris crucifer), American toads (Bufo 

americanus), green frogs (Rana clamitans) and wood frogs (Rana sylvatica) was studied 

at early (Gosner stages 25-27) and late (stages 29-36) developmental stages. The results 

of the study showed counterintuitive patterns of survivorship; survival was significantly 

lower for all animals exposed to 3 ppb compared with either 30 or 100 ppb, except the 

late stages of the toad and wood frog. There were significant differences in survivorship 

between the 3 ppb treatment group and the control for the late stage of the American toad 

and both developmental stages of the green frog. However, no significant differences in 

 

59 Rohr, J. R., T. M. Sesterhenn and C. Stieha. 2011. Will climate change reduce the effects of a pesticide on 

amphibians?: partitioning effects on exposure and susceptibility to contaminants. Global Change Biology. 17: 657 

- 666 (ECOTOX Ref. No. 153803) 

60 Storrs, S.I. and J.M. Kiesecker. 2004. Survivorship Patterns of Larval Amphibians Exposed to Low 

Concentrations of Atrazine. Environ. Health. Perspec. 112(10). p. 1054-1057 
35

survivorship, relative to the control, were observed for late spring peepers, early 

American toads, and late wood frogs. 

Rohr et al. 200361examined the effects of atrazine on streamside salamanders 

(Ambystoma barbouri), and No effect on embryo or larval survival, hatching, or growth 

or development (i.e., mass, SVL, and limb deformities) rates were observed at ≥400 

μg/L. , Systematically tapping tanks caused a greater activity in larvae exposed to 400 

μg/L atrazine, suggesting underlying nervous system malfunction according to the study 

author. Hunger stimulated a decrease in refuge use and an increase in activity (based on 

regression; no significant difference detected at any level); however this response was 

least pronounced in the larvae exposed to atrazine at 400 μg/L, possibly because these 

larvae were the most lethargic. The study authors speculated that the frog collection site 

may have been influenced by agricultural activity including atrazine. In addition, DMSO 

was used as a solvent at a concentration greater than recommended for chronic testing. 

In Rohr et al. 200462, the combined effects of atrazine, food limitation, and drying 

conditions on the survival, life history, and behavior of the streamside salamander 

(Ambystoma barbouri) from the embryo stage through metamorphosis were examined. 

Exposure to 400 μg/L atrazine decreased embryo survival to Day 16 and increased time 

to hatching. However, most embryo mortality was associated with a white film covering 

the embryo, suggesting the presence of a fungal pathogen. It is unknown whether the 

fungi caused or simply followed mortality. Drying conditions and food limitation 

decreased larval survival, while 400 μg/L atrazine only reduced larval survival in one of 

the two years tested. Drying conditions accelerated metamorphosis for larvae exposed to 

0 and 4 μg/L atrazine, but did not affect metamorphosis timing for the 40 or 400 μg/L 

groups. The study author states that effects on metamorphosis were affected at 40 μg/L. 

Therefore the overall NOAEC was 4 μg/L. DMSO was used as the solvent and a 

negative control was not tested. Frog collection site may have been influenced by 

agricultural activity (based on Rohr et al. 2003) which included atrazine. 

An examination of water conserving behavior was examined in A. barbouri salamanders, 

Rohr and Palmer 200563. This study was a continuation of Rohr et al. 2004, and 

metamorphs used in this study which were obtained from that study and were not further 

exposed to atrazine in this study. For every hour for 5 hours the mass of the metamorphs 

was recorded and they were observed for water-conserving behaviors (moving and 

pressed against dish and number of organisms huddled (when dished contained 3 

metamorphs)). The same method description above was used to retest the same animals a 

mean of 237 and 239 days after atrazine exposure. According to the authors, the 

salamanders exposed to 40 and 400 μg/L exhibited greater activity and fewer water-

 

61 Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. Niedzwiecki, T. Sager, A. 

Sih, and B.D. Palmer. 2003. Lethal and Sublethal Effects of Atrazine, Carbaryl, Endosulfan, and 

Octylphenol on the Streamside Salamander (Ambystoma barbouri). Environmental Toxicology and 

Chemistry, Vol. 22, No. 10. p. 2385-2392 

62 Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. Niedzwiecki, T. Sagar, A. 

Sih, and B.D. Palmer. 2004. Multiple Stressors and Salamanders: Effects of an Herbicide, Food 

Limitation, and Hydroperiod. Ecological Applications. 14(4). p. 1028-1040 

63 Rohr, J.R. and B.D. Palmer. 2005. Aquatic herbicide exposure increases salamander desiccation risk 

eight months later in a terrestrial environment. Environ. Toxicol. Chem. 24(5): 1253-1258 
36

conserving behaviors and accelerated water loss compared to the control for both the four 

and eight months post atrazine exposure. As this study was a continuation of Rohr et al. 

2004, the same uncertainties as identified in that study are expressed here. In addition, 

for this trial, the authors state that percent mass loss was influenced by placement in the 

test system. 

Streamside salamander Ambystoma barbouri) embryos and larvae were exposed to 

atrazine (from Rohr et al. 2004) and survival was quantified during and 14 months after 

exposure (Rohr et al. 2006)64. Atrazine-induced mortality during exposure was 

ameliorated by density-dependent survival after exposure, but complete densitydependent-mediated compensation was precluded by significant “carryover effects” of 

atrazine. Salamanders exposed to 4, 40 and 400 μg/L (all treatment groups) of atrazine 

had significantly lower survival than did control animals 14 months (421 days) 

postexposure. Again, as the source of organism for this study is from Rohr et al. 2004, 

the same concerns apply to this study. 

In a study by Olivier and Moon 201065, the effects of atrazine on spotted salamander 

(Ambystoma maculatum) eggs and their symbiotic algae was examined. Eggs (Harrison 

Stage 10 – 17) were exposed to atrazine concentrations of 50, 100, 200 and 400 μg/L for 

67 days and algal growth within the eggs was scored based on their green color intensity. 

However, there are issues with this study that limit its utility. Aggregate egg masses 

were assessed it is uncertain whether the study could accurately determine the number of 

embryos within large three dimensional masses. Also, the climate control system failed 

briefly during the study and soon afterward there was a 14 – 20% increase in egg mass 

pathologies during this time across all study groups. Egg mass mortalities abruptly 

increased at this time as well. 

In a mesocosm study, a decrease in amphibian length and weight at metamorphosis at 

200 μg/L, NOAEC 20 μg/L, was reported for Hyla versicolor (Diana et al. 2000)66. It 

appears that atrazine was detected in the control samples at about 8% of the lowest test 

concentration. 

In another mesocosm study by Boone and Bridges-Britton 200667, the single and 

interactive effects of atrazine, carbaryl, and ammonium nitrate fertilizer on 

metamorphosis of tadpoles of the gray treefrog (Hyla versicolor) was examined. The 

interaction of the atrazine and ammonium nitrate fertilizer marginally influenced survival 

 

64 Rohr, J.R., T. Sager, T.M. Sesterhenn, and B.D. Palmer. 2006. Exposure, Postexposure, and DensityMediated Effects of Atrazine on Amphibians: Breaking Down Net Effects Into Their Parts. Environ. 

Health Perspec. 114(1). p. 46-50. 

65 Olivier, H. M. and R. R. Moon. 2010. The effects of atrazine on spotted salamander embryos and their symbiotic 

alga. Ecotoxicology 19: 654 – 661. (ECOTOX Ref No. 153629) 

66 Diana, S. G., Resetarits, W. J. Jr., Schaeffer, D. J., Beckmen, K. B., and Beasley, V. R. (2000). Effects of 

Atrazine on Amphibian Growth and Survival in Artificial Aquatic Communities. Environ.Toxicol.Chem.

19: 2961-2967 

67 Boone, M.D. and C.M. Bridges-Britton. 2006. Examining multiple sublethal contaminants on the gray 

treefrog (Hyla versicolor): effects of an insecticide, herbicide and fertilizer. Environ. Toxicol. Chem. 

25(12): 3261-3265 
37

to metamorphosis (p= 0.0559) The results of the study indicated that the presence of 

multiple, sublethal chemical stressors with different modes of action may not be more 

detrimental than that of one chemical factor alone. However, the power of the study to 

detect differences was reported as low. 

In the mesocosm study by Du Preez et al. 2008, which was a continuation of Jooste et al. 

2005, X. laeavis metamorphs were evaluated over a multigeneration with observations on 

breeding. The authors state that there was no effect on clutch size, hatching success, 

growth or sexual development in the study at 25 μg/L. They authors also state that the 

size of the females influenced the clutch size with large differences in number of eggs. 

As this is a continuation of Jooste et al. 2005, the uncertainties identified in this study 

(from the 2007 SAP on amphibians) are of concern in this study. In that study 

uncertainties identified included: unusually long development times; feeding and 

temperatures may have limited development and higher incidence of testicular oocytes in 

controls. 

Several studies examined acute mortality to the embryo-larval stage of the amphibians. 

In Howe et al. 199868 96 hour LC50 values for the northern leopard frog (Rana pipiens) 

and American toad (Bufo americanus) were 14,500 and 10,700 μg/L, respectively. 

However raw data was not provided for this study and the study was conducted using a 

formulation (40.8% atrazine), therefore, there is uncertainty in if the observed toxicity 

was due to atrazine, some other component in the formulation or a combination of both 

For Birge et al. 198069 reported 4 days post hatch LC50 values for four different frog 

species ranged from 410 ->48, 000μg/L. The LC50 values were a combination of 

mortality and abnormalities that are assumed to result in lethality. In a assays that was 

conducted under the ASTM frog embryo teratogenic assay-Xenopus (FETAX), the acute 

LC and EC50 values for Xenopus laevis and X. tropicalis were ≥2,100 μg/L. However, 

this assay employs the use of a non-recommended solvent (DMSO) and at higher than 

recommended concentration (1% in this assay as oppose to maximum rate of 0.05%) and 

greater loading than recommended. In Wan et al. 200670, acute toxicity was examined 

using technical atrazine and a 48.5% formulation for the tadpole Rana catesbeiana (10 

cm in length). The 96-hr LC50 value for technical and the atrazine formulation was 

>16,000 μg/L and > 480,000μg/L (not ai). A 28-day sediment study with atrazine and 

Hyalella azteca did not report any effects on survival or dry weight at 0.1 mg/kg. 

 

68 Howe, G.E., R. Gillis, and R.C. Mowbray (1998). Effect of chemical synergy and larval stage on the 

toxicity of atrazine and alachlor to amphibian larvae. Environmental Toxicology and Chemistry, 17(3): 

519-525 

69 Birge, W. J., J. A. Black and R. A. Kuehne. 1980. Effects of organic compounds on amphibian 

reproduction. University of Kentucky, Water Resour. Res. Inst., Res. Rep. 121. 39 p. (USDI, Agreement 

Numbers: 14-34-0001-7038 (FY 1977), 14-34-0001-8019 (FY 1978), and 14-34-0001-9091 (FY 1979). 

(MRID # 452083-02).

70 Wan M.T., C. Buday, G. Schroeder, J. Kuo, and J. Pasternak. 2006. Toxicity to Daphnia magna, 

Hyalella azteca, Oncorhynchus kisutch, Oncorhynchus mykiss, Oncorhynchus tshawytscha, and Rana 

catesbeiana of Atrazine, Metolachlor, Simazine, and Their Formulated Products. Bull. Environ. Contam. 

Toxicol. 76: 52-58 
38

The genotoxic potential for Rana catesbeiana species was examined in Clements et al. 

199771 and the study reported that all atrazine concentrations ≥4810 μg/L had significant 

increases in DNA ratios relative to control and the linear correlation between DNA 

damage and atrazine concentrations was moderate; all animals died at 308,000μg/L. This 

study was also conducted with a formulation (88.2%), therefore there is uncertainty in the 

source of toxicity and the study was lacking in details on study design such as number of 

replicates and number of organisms exposed. In Freeman and Rayburn 200472 American 

toads (Bufo americanus) and Xenopus laevis tadpoles were exposed to atrazine and DNA 

from tadpoles was examined. According to the study authors, atrazine did not appear to 

be genotoxic for both species at atrazine concentrations of up to 10,000 μg/L. Water 

quality parameters not reported in this study and unsure if a solvent was used. In another 

study, Freeman et al. 200573, American toads (Bufo americanus) were also exposed to 

atrazine and DNA from tadpoles was examined. The total number of nuclei were not 

significantly different; however, control tadpoles at significantly earlier stage and 

weighted less than tadpoles at 800 μg/L atrazine; in the second experiment there were no 

effects on stage, weight or number of nuclei and in a third experiment tadpoles at 

atrazine concentrations of 250, 500 and 1000 μg/L, but not 5000 and 10000 μg/L, were at 

a significantly greater stage than then controls. In this study, water quality parameters 

were not reported and as stage and growth are correlated it is unsure how organisms at 

different stages was accounted for when analyzing mass. 

Several studies were reported by Lenkowski et al. and Lenkowski and McLaughin 

(2008, 2010a and 2010b)74 in which either early-stage X. laevis or Bufo americanus 

tadpoles were examined for atrazine effects on organogenesis. These studies reported 

effects at relatively high test concentrations ≥10,000 μg/L. For all of the studies, DMSO 

was used at concentrations higher than recommended (0.1-0.35 % vs recommended 

0.05%). For Lenkowksi et al. 2008, the study authors stated that malformations of the 

heart and visceral hemorrhaging and that atrazine significantly increased heart 

malformations across all treatments (10-35 mg/L). Gut malformations and edema were 

also significant at all treatment groups. Axis malformations were significant at 25 and 35 

mg/L. In Lenkowski and McLaughlin 2010, The study authors state that at 35 mg/L, 

acute atrazine exposure disrupted the structural integrity of differentiated cartilage and 

muscle and reduced and distorted cranial cartilage and matrix metalloproteinases that the 

authors state has described roles during X. laevis development were affected at 3.5-35 

 

71 Clements, C., S. Ralph, and M. Petras. 1997. Genotoxicity of select herbicides in Rana catesbeiana 

tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay. Environ. Mol. Muta. 

29:277-288 

72 Freeman J.J. and A.L. Rayburn. 2004. In vivo genotoxicity of atrazine to anuran larvae. Mut. Res. 560: 

69-78.

73 Freeman J.L. N. Beccue and A.L. Rayburn. 2005. Differential metamorphosis alters the endocrine 

response in anuran larvae exposed to T3 and atrazine. Aqua. Tox. 75: 263-276 74 Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin. 2008. Perturbation of organogenesis 

by the herbicide atrazine in the amphibian Xenopus laevis. Environ. Health Perspect. 116 (2): 223-230; 

Lenkowski, J.R. and K. A. McLaughlin. 2010. Acute atrazine exposure disrupts matrix metalloproteinases 

and retinoin signaling during organ morphogenesis in Xenopus laevis. J. App. Toxicol. 30: 582-589; 

Lenkowski, J.R, G.Sanchez-Bravo, K.A. McLaughlin. 2010. Low concentration of atrazine, glyphosate, 

2,4-dischlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ 

morphogenesis. J. Environ. Sci. 22(9): 1305-1308. 
39

mg/L. For Lenkowski et al. 2010b, intestinal malformations and edemas were 

significantly increased in the 3.5, 10 and 35 mg/L atrazine concentrations. Also, 

according to the study authors, the cranial muscles were reduced in the 35 mg/L atrazine 

concentration compared to the non-treated controls. 

Rana catesbeiana tadpoles were exposed to atrazine to examine whether atrazine and its 

degradate, desethylatrazine (DEA) alters the steady-state levels of SF-1 and CYP19 

cDNA transcripts in the brain of premetamorphic tadpoles (Gunderson et al. 2011)75. ). 

No effect on SF-1 transcript was observed for atrazine or DEA treatment. A significant 

positive correlation between CYP19 and SF-1 transcript abundance was observed in the 

atrazine treatment (correlation coefficient 0.526, p=0.017) but was not observed for DEA. 

However, no negative control was included and the solvent (isopropyl alcohol) is not 

recommended. 

 

75 Gunderson, M.P, N. Veldhoen, R.C. Skirrow, M.K. Macnab, W. Ding, G. van Aggelen, and C.C. 

Helbing. 2011. Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome 

P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles 

(Rana catesbeiana). Aquat. Toxicol. 102: 31-38 
40

3. INDIVIDUAL OPEN LITERATURE SUMMARIES (in alphabetic order) 

3.1. Allran, J.W., W.H. Karasov. 2000 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 56572 

Allran, J.W., W.H. Karasov. 2000. Effects of atrazine and nitrate on northern leopard 

frog (Rana pipiens) larvase exposed in the laboratory from posthatch through 

metamorphosis. Environ. Tox. Chem. 19(11): 2850-2855. ECOTOX 56572 

Purpose of Review: Registration Review

Date of Assessment: 03/05/12 

Brief Summary of Study Findings: 

The objective of the study was to determine whether atrazine and nitrate (alone and in 

combination) affected northern leopard frogs from posthatch through metamorphosis. 

Methods 

Rana pipiens larvae (Gosner (G) stage 25; received as eggs from 4 different clutches 

from Nasco (Fort Atkinson, WI)) were exposed to three atrazine (99%) concentrations (0, 

20, and 200 μg/L) and three nitrate concentrations (0, 5, 30 NO3-N/L) for a total of nine 

treatments each with 4 replicates with 20 larvae per replicate (2.5 gal aquaria with 7-L 

solution). Dechlorinated water used as dilution water and aquaria aerated; a solvent did 

not appear to be used. Aquaria placed in water bath at 22°C and solutions renewed every 

48 hours. Larvae fed mixture of boiled, pureed romaine lettuce, rabbit chow, fish flakes at 

a nonlimiting rate. Atrazine analytical water samples collected weekly. Every 7 days, 10 

randomly selected larvae/tank length and stage were measured. At metamorphosis 

(G42), frogs transferred to another aquaria with 1L treatment solution until tail resorption 

after which juveniles anesthetized and mass measured and blood drawn from heart and 

hematocrit determined. Experiment terminated after 138 d when 90% of tadpoles 

completed metamorphosis (60 of initial 720 didn’t complete metamorphosis). Atrazine 

residues were measured in frog tissue; blood not taken from frogs used for tissue 

analysis. 

Growth and development data were analyzed by randomly selecting the results from 10 

of the 20 exposed tadpoles per tank. All other endpoints analyzed using mean values 

from all individuals. 
41

Results 

Measured atrazine concentrations were 19.19 and 192 μg/L for the 20 and 200 μg/L, 

respectively [measurements in control solution not reported]. Nitrate concentrations were 

5.4 and 29.2 mg/L for the 5 and 30 mg/L groups, respectively. According to the study 

authors, atrazine, nitrate and their interaction had no significant effect on percent 

metamorphosis, time to metamorphosis, percent survival, mass at metamorphosis or 

hematocrit (Table 1). 

Nitrate did significantly decrease larval length (p=0.0190), but according to the study 

author they were never more than a couple of millimeters in difference While there was 

no consistent effect of atrazine on growth as a significant atrazine by time effect was 

observed (p=0.0001) and a significant atrazine-by-nitrate-by time effect (p=0.0107) 

(Figure 1). There was also no consistent effect of atrazine or nitrate on development; the 

authors indicated a significant atrazine-by-nitrate-by-time effect was observed and 

clutches differed significantly only for developmental stage (p=0.0082) (Figure 2). 

Atrazine concentrations in juveniles were 128 μg/L for the 20 μg/L group (n=12 aquaria) 

and 1,147 μg/L for the 200 μg/L group (n=12 aquaria); indicating a bioconcentration 

factor of ca. 6 times. 
42

Description of Use in Document: 

Qualitative. 

Rationale for Use: Provides information 

about the growth and development of the northern leopard frog, however there are some 

uncertainties in the reporting of the results. 

Limitations of the Study: The results of the growth and development endpoints were 

based on a subset of the data. It is uncertain of what the results would have been, for the 

results at test termination, if the entire data set had been included. Although the study 

author reported that there was no consistent effect on growth for atrazine (Table 1) they 

did indicate a significant atrazine by time effect; however the authors did not state for 

which concentration at which time points. As the tadpoles were going through 

metamorphosis during the study which impacts the length and weight of a tadpole it is 

uncertain of how they accounted for potential different stages affecting the growth 

parameters. 

Data on survival is not presented, therefore, control survival is not known. The authors 

state that more than 90% of the animals reached metamorphosis with one to two larvae 

remaining in each tank. Therefore, it could be inferred that the mortality was <10% 

across the treatments. The egg clutches were retained and tested separately (within a 

replicate) rather than combining all four clutches and indiscriminately distributing larvae. 

It is not known why the clutches were kept separate or what the impact would have been 

if they had been mixed. Summary data for the treatment groups for the hematocrit 

endpoint are not reported. For all reported endpoints, all replicates (n=36) were 

combined to give one mean and SE which was reported in Table 1 in report. 

It is not known if the adults used to produce the eggs used in the study were laboratory 

raised or field collected as prior exposure history is not reported. Chlorine levels in the 

dechlorinated water are not reported as the analysis for potential contaminants in the feed 

and water are not reported. 
43

Reviewer: Amy Blankinship, Chemist, Environment Risk Branch 3, Environmental Fate 

and Effect Division. 

 
44

3.2. Allran J.W and W.H. Karasov. 2001 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 59251 

Allran J.W and W.H. Karasov. 2001. Effects of atrazine on embryos, larvae, and adults 

of anuran amphibians. Environ. Toxicol chem. 20(4): 769-775. ECOTOX 59251 

Purpose of Review: Litigation

Date of Assessment: 5/10/06 

Brief Summary of Study Findings: 

Adult Rana pipiens frogs (N=4/group) were exposed to seven atrzine concentrations 

ranging from 1.6 – 20 mg/L for 14 days, and ventiliation rates, feeding, and hemoglobin 

levels were evaluated. 

In a separate study, 30 embryos were exposed to 6 atrazine concentrations ranging from 

2.6 to 20 mg/L under static-renewal conditions, and hatchability (Day6) and post hatch 

effects including mortality, deformity, and swimming speed were evaluated 96-hours 

after hatching for amphibian species (R. pipiens, R. sylvatica, and Bufo americanus). 

The results experiments on adult frogs suggest that buccal ventilation (breaths/minute) 

was significantly (p<0.05) at 4.3 mg/L and higher, and thoracic ventilation was 

significantly (p<0.05) increased at 12 mg/L and higher. Feeding was reduced at 20 mg/L. 

Embryos exposed to atrazine at 4.3 mg/L and higher were observed with a significant 

(p<0.05) increase in deformities, but did not affect hatchability or 96-hr post hatch 

mortality, 

Description of Use in Document: Qualitative. 

Rationale for Use: Amphibian data that may provide insight into effect levels into adult 

and embryo exposures. 

Limitations of the Study: 

Raw data were not provided. Low levels of atrazine were observed in the frog collection 

site (up to 0.06 ug/L). These background levels were considered “negligible” by the 

study authors. 

Blood was collected before 7 or 8 days before atrazine treatment (assuming via cardiac 

punch under anesthesia); it is unknown how this additional handling and potential stress 

impacted the animals. 

Reviewer: Brian Anderson, ERB3 
45

3.3. Birge, W. J., J. A. Black and R. A. Kuehne. 1980 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 6187 

Birge, W. J., J. A. Black and R. A. Kuehne. 1980. Effects of organic compounds on 

amphibian reproduction. University of Kentucky, Water Resour. Res. Inst., Res. 

Rep. 121. 39 p. (USDI, Agreement Numbers: 14-34-0001-7038 (FY 1977), 14-

34-0001-8019 (FY 1978), and 14-34-0001-9091 (FY 1979). (MRID # 452083-

02). 

. 

Purpose of Review: Registration review

Date of Assessment: 5/01/12 

Brief Summary of Study Findings: 

Species used were Bufo americanus (American toad), Rana catesbeiana (bullfrog), Rana 

palustris (pickerel frog) and Rana pipiens (northern leopard frog); frogs were either 

collected from Frankfort National Fish Hatchery, Frankfort Kentucky or obtained from 

Mogul-Ed, Oshkosh WI or Charles Sullivan, Nashville TN. Atrazine was tested using a 

80% wettable powder (formulation not stated) and test solutions were confirmed daily. 

Tests were conducted under flow-through conditions, 2.5 hr/turnover. Embryos were 

tested at 30 minutes after fertilization to 2-6 hours postspawning. Sample sizes per test 

concentration ranged from 50-130 eggs and eggs were examined daily and dead embryos 

removed. Temperature, dissolved oxygen, water hardness, pH, and specific conductivity 

was monitored in the test water. Percent survival of normal organisms (free of gross 

teratic defects) was determined at hatch and 4 days post hatch. 

LC50 results are presented in table below. These values represent organisms that died as 

well as organisms with gross teratogenic effects. 
46

Description of Use in Document: Qualitative. 

Rationale for Use: ,Provides information about the teratogenic potential of atrazine on 

amphibian embryos and young larvae. 

Limitations of the Study: Solvent (if used) was not identified. As formulation used, 

uncertainty in source of toxicity (technical atrazine, inert ingredient or some 

combination). LC50s were not based on mortality per se, but on abnormalities that would 

reportedly preclude survival under natural conditions. 

Reviewer: Amy Blankinship, ERB3 

 
47

3.4. Boone, M.D. and C.M. Bridges-Britton. 2006 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 88290 

Boone, M.D. and C.M. Bridges-Britton. 2006. Examining multiple sublethal 

contaminants on the gray treefrog (Hyla versicolor): effects of an insecticide, herbicide 

and fertilizer. Environ. Toxicol. Chem. 25(12): 3261-3265. 

Purpose of Review: Registration Review

Date of Assessment: 5/1/12 

Brief Summary of Study Findings: 

This study examined the single and interactive effects of atrazine, carbaryl, and 

ammonium nitrate fertilizer on metamorphosis of tadpoles of the gray treefrog (Hyla 

versicolor). Tadpoles were reared in mesocosms from hatching through metamorphosis 

and were exposed to the presence or absence of the three contaminants. The results of 

the study indicated that the presence of multiple, sublethal chemical stressors with 

different modes of action may not be more detrimental than that of one chemical factor 

alone. 

Methods 

Eggs field collected and hatched in the laboratory and held until Gosner stage 25. 

Mesocosms were established in 24 polyethylene ponds with a screen mesh lid. Three 

factors used in the study (carbaryl (0 or 2 ppm), ammonium nitrate fertilizer (0 or 10 

ppm) and atrazine (0 or 20 ppb)) (3 replicates/treatment). Thirty gray treefrogs were 

added to each pond and were acclimated for 8 days prior to addition of chemicals, 

atrazine was Aatrex (40.8%). Water quality and periphyton periodically measured and 

metamorphs removed from ponds daily and held in laboratory until tail resorption at 

which time they were weighed. Study terminated on Day 35 and ponds drained to search 

for remaining larvae. 

Total surviving larvae (larvae and metamorphs) was 83.75%. The interaction of the 

atrazine and ammonium nitrate fertilizer marginally influenced survival to 

metamorphosis (p= 0.0559) (see figure below). No other effects with atrazine were 

reported. 
48

Description of Use in Document: Qualitative. 

Rationale for Use: Provides information about effects from atrazine and fertilizer 

interaction but there are uncertainties in study. 

Limitations of the Study: 

Low power to detect differences. 

Reviewer: Amy Blankinship, ERB3 

 
49

3.5. Boone, M. D. and S. M. James. 2003 

Open Literature Review Summary 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Record Number and Citation: 

Boone, M. D. and S. M. James. 2003. Interactions of an insecticide, herbicide, and 

natural stressors in amphibian community mesocosms. Ecological Applications. 13(3): 

829-841. (MRID 48261104) 

Purpose of Review: 

Determine potential effects of atrazine on an aquatic community. Endpoints from this 

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant 

communities and associated determination of a Level of Concern (LOC). 

Date of Review: September 4, 2009

Purpose of Study: 

This study examines the effects of atrazine and carbaryl (study design included individual 

and combined exposure treatments) on phytoplankton and four amphibian species in 

outdoor mesocosms. Tadpole density and photoperiod were also manipulated.

Summary of Study Findings: 

The focus of the summary is on atrazine treatments (treatments including carbaryl are not 

reported). 

Methods: 

Egg masses were collected for southern leopard frogs (Rana sphenocephala; 3 egg 

masses) and spotted salamanders (Ambystoma maculatum; 21 egg masses) on March 14, 

2000 and March 27, 2000, respectively, from the Basket Wildlife Area in Boone County, 

Ashland, Missouri. Egg masses were also collected for American toads (Bufo 

americanas; 7 egg masses) and small-mouth salamanders (Ambystoma texanum; ca. 30 

egg masses) on March 30, 200 from the Forum Nature Area in Boone County, Columbia, 

Missouri and April 9, 2001 from the Basket Wildlife Area, respectively. Eggs were 

hatched in the laboratory at 23-25°C and held until all larvae were free-swimming. 

Clutches were mixed within each species to homogenize genetic variation. 
50

The aquatic communities were prepared by filling polyethylene cattle tank ponds (1.85 m 

in diameter, 1480 L volume) with 1000 L of tap water, 1 kg of leaf litter from a mixed, 

deciduous forest and plankton from natural ponds (500 mL of plankton per pond at six 

different times) in mid-March 200 and 2001. The plankton populations were wellestablished prior to addition of larvae. The aquatic communities were maintained 

outdoors during the experiments to simulate natural conditions. In order to protect the 

test systems, the cattle pond tanks were located inside a fenced field and covered with 

mesh lids during the experiments. 

For Experiment I, the effects of competition, atrazine and carbaryl were studied by a fully 

crossed design with three replicates (36 ponds) where initial anuran density was either 

low (20 tadpoles/1000 L) or high (60 tadpoles/1000 L), carbaryl concentration was 0, 3.5 

or 7.0 mg/L, and atrazine concentration was 0 or 200 μg/L. Twelve spotted salamanders 

were also in each pond. Carbaryl (liquid Sevin, 21.3% carbaryl) and atrazine (liquid 

Aatrex, 40.8% atrazine) were added via mixing with 5 L of pond water and pouring 

evenly over the pond surface without stirring. Uncontaminated pond water (5 L) was 

added in the same manner to each control pond. The herbicide/insecticide treatment was 

added between 1000 and 1100 hours Central Standard Time (pond water: pH 7.9 ± 0.01, 

14.6 ± 0.06°C). Ponds with only carbaryl were sampled (triplicate 2-L aliquots from 

each of the three ponds) at 1, 24, 48 and 96 hours post-treatment. Samples from each 

pond were combined, and an aliquot (20 mL) was refrigerated for analysis. Ponds with 

only atrazine were sampled (triplicate 2-L aliquots from each of the three ponds) at 1, 15 

and 57 days post-treatment. Samples from each pond were combined, and an aliquot (1 

L) was refrigerated for analysis. The half-lives were determined after HPLC analysis via 

linear regression of the determined amounts: ca. 4.5 days for carbaryl and 34 days for 

atrazine. After 56-58 days (39-41 days of exposure), the experiment was terminated. 

Most of the tadpoles and salamander larvae had not reached metamorphosis. Mean body 

mass, developmental stage, snout-vent length (SVL, salamander larvae only) and pond 

survival was determined. Data were analyzed for effects of carbaryl, atrazine, density 

(for tadpoles) and all interactions on mass, developmental stage and SVL using 

multivariate analysis of covariance (MANCOVA) for ponds with both species. 

Univariate analysis of covariance (ANCOVA) was used for determining which responses 

were the strongest contributors to the multivariate effects. Survival was analyzed using 

analysis of variance (ANOVA). ANOVA was also used to determine differences in the 

control ponds with one or both species. To normalize data and stabilize variances, the 

study authors angularly transformed all proportion data, log transformed mass and SVL, 

and used rank procedure (PROC RANK) on developmental stage data before analyses. 

Conservative pair-wise comparisons for significant main effects were performed using 

Scheffé’s multiple comparison tests. 

For Experiment II, the effects of hydroperiod, atrazine, and carbaryl on amphibians 

reared through metamorphosis were studied by a fully crossed design with four replicates 

(32 ponds) where hydroperiod was either constant or dried with a movable standpipe, 
51

carbaryl concentration was 0 or 5.0 mg/L, and atrazine concentration was 0 or 200 μg/L. 

Twelve small-mouthed salamanders and 45 American toads were also in each pond; 

initial anuran density was not variable since the results of the first experiment indicated 

that competition between anurans and salamanders had no effect. Carbaryl (liquid Sevin, 

22.5% carbaryl) and atrazine (liquid Aatrex, 40.8% atrazine) were added. The 

herbicide/insecticide treatment was on day 8 (pond water: pH 7.7 ± 0.03, 13.3 ± 0.04°C). 

Ponds with only carbaryl were sampled (triplicate 2-L aliquots from each of the three 

ponds) at 1 and 48 hours post-treatment. Samples from each pond were combined, and 

an aliquot (20 mL) was refrigerated for analysis. Ponds with only atrazine were sampled 

(triplicate 2-L aliquots from each of the three ponds) at 1 day post-treatment. Samples 

from each pond were combined, and an aliquot (1 L) was refrigerated for analysis. 

Atrazine concentration was confirmed as 197 ug/L. After 88 days (80 days of exposure), 

the experiment was terminated. Mean body mass, time to metamorphosis, and pond 

survival was determined. Toad data were analyzed for effects of carbaryl, atrazine, 

hydroperiod and all interactions on mass and time to metamorphosis using MANCOVA; 

survival was the covariant. ANCOVA was used for determining which responses were 

the strongest contributors to the multivariate effects. Survival was analyzed using 

analysis of variance (ANOVA). Salamander data were analyzed for effects of carbaryl, 

atrazine, hydroperiod, carbaryl by hydroperiod, and atrazine by hydroperiod interactions 

on mass and time to metamorphosis using MANCOVA; survival was the covariant. 

ANCOVA was used for determining which responses were the strongest contributors to 

the multivariate effects. Prior to analysis, the following transformations were performed: 

angular transformation (all proportion data) and log transformation (mass and time to 

metamorphosis). 

Chlorophyll content was measured by taking a 1-L composite sample (from three 2-L 

samples) from each pond. An aliquot (100 mL) was filtered and incubated with 15 mL of 

neutralized 90% acetone in the dark at 5°C for 24 hours. Chlorophyll was measured 

using fluorometry. Water samples were taken before chemical application on day 15 and 

then on days 22, 29, and 42 (all post-application) for Experiment I. For experiment II, 

water samples were taken before chemical application on day 8 and then on day 50 (42 

days of exposure). Data were analyzed for effects of time, carbaryl, and atrazine for both 

experiments in addition to density (Experiment I) or hydroperiod (Experiment II) using 

ANCOVA. 

Results: 

 Experiment I: effects of competition, atrazine, and carbaryl on larval amphibians 

Atrazine exposure at 200 ug/L did not influence the mass, SVL, developmental stage, or 

survival of spotted salamanders. There was a significant interaction of atrazine and 

carbaryl on SVL that the authors attributed to atrazine acting as an ameliorating agent for 

the carbaryl treatments. Similar observations occurred on body mass and developmental 

stage, although the interaction was not significant (P>0.05). For leopard frogs, atrazine 

exposure did not significantly influence survival. However, survival was significantly 
52

influenced by an atrazine by population density interaction where atrazine exposure 

decreased survival in the high-density ponds but not the low-density ponds relative to the 

control ponds (treatments without atrazine). The multivariate responses of leopard frogs 

were significantly affected by atrazine exposure; mass significantly decreased with 

atrazine exposure compared to the controls (P=0.0052). Results of the competitive 

controls indicated that no difference occurred between spotted salamanders reared alone 

or with southern leopard frogs. 

 Experiment II: effects of hydroperiod, atrazine, and carbaryl on amphibians reared 

through metamorphosis 

Multivariate analyses on salamanders indicated that atrazine exposure and hydroperiod 

(individually and their interaction) significantly affected mass and time to 

metamorphosis. The effect of atrazine exposure was attributed to increasing time to 

metamorphosis. Time and mass to metamorphosis were affected by the atrazinehydroperiod interaction, resulting in longer larval periods in constant hydroperiods and 

smaller mass at metamorphosis in drying hydroperiods. For American toads, multivariate 

responses were significantly affected by atrazine exposure. Atrazine exposure 

significantly reduced toad mass at metamorphosis. 

 Chlorophyll content 

In Experiment I, atrazine exposure had a significant effect on chlorophyll content. After 

12 days of exposure, significantly lower chlorophyll content was observed in ponds with 

atrazine exposure of 200 μg/L (1.31 ± 0.48 μg/L) versus the control ponds (3.88 ± 0.48 

μg/L; F1,24 = 15.65, P = 0.0006). Examination of Figure 1 suggests that the chlorophyll 

content (3.88 μg/L) reported by the authors for control ponds included all ponds without 

atrazine (i.e., some ponds contained carbaryl). The authors state that there was no 

difference between treatments and controls on day 42 (after 25 days of exposure); 

however, it was not stated if ‘no difference’ meant lack of a statistically significant 

difference. However, chlorophyll content was higher than controls after 25 days of 

exposure (day 42; See Figure 1 below). 

In Experiment II, the effects of atrazine exposure on chlorophyll content were not 

discussed, but Table 5 (summary in Table 1 below) of the study report indicates that 

atrazine did not have significant effects on chlorophyll at the end of the study (P = 0.62). 

However, chlorophyll content was only measured after 42 days of exposure in 

experiment II. In experiment I, chlorophyll content had already recovered after 25 days 

of exposure so it is reasonable to assume that any effect in experiment II may have been 

missed due to insufficient sampling. No figures were provided which illustrated the 

chlorophyll content through Experiment II. 
53

Table 1. Summary of repeated-measures ANOVA for effects of time and atrazine exposure on 

chlorophyll content in pond water in Experiments I and II (from Table 5 in Boone and James, 2003). 

Source of variation 

Wilks’ 

lambda df F P 

Experiment I 

 Time 0.0351 3, 22 201.69 0.0001 

 Time × atrazine 0.6858 3, 22 3.36 0.0371 

Experiment II 

 Time 0.1974 1, 24 97.59 0.0001 

 Time × atrazine 0.9895 1, 24 0.25 0.6190 

Figure 1. Pond chlorophyll measures for an atrazine-by-carbaryl interaction over time from 

Experiment I. Numbers represent the concentration of carbaryl (in milligrams per liter) and atrazine 

(in micrograms per liter), respectively, to which the ponds were originally exposed (Figure 10 in 

Boone and James, 2003). 
54

Data Summary Table: 

Effect 

Magnitude 

of Effect 

relative to 

controls 

Nominal 

Atrazine 

Concentration 

(μg/L) 

Day of Study 

Effect Occurred 

Time 

Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Reduction in 

chlorophyll 

content 

None 2001 Not applicable Not applicable Not 

applicable 

Control levels were 

higher than all 

treatment levels at 

Day 12 in 

Experiment 1 

(Figure 1). 

However, there 

were no apparent 

decreases in 

chlorophyll in 

other treatment 

levels with atrazine 

and chlorophyll in 

the atrazine-only 

treatment was 

higher that the 

control on Day 25. 

There were no 

apparent effects in 

Experiment 2 

(Table 1) although 

this analysis 

appears to have 

included treatments 

containing both 

atrazine and 

carbaryl, thus is not 

considered for the 

summary table (see 

reviewer 

comments). 

No experimental 

variability was 

available for this 

effect. 1

 During experiment 1, atrazine was measured at day 1 (207 ug/L) following exposure. 

Reviewer Comments: 

The study was mainly an amphibian study in which chlorophyll content was measured in 

order to examine effects of atrazine on amphibians via diminishing their food source, the 

aquatic plant life. The study authors were looking for statistical trends and relationships, 

and a limited amount of the data used in the analyses was reported. 
55

The study authors reported that previous studies by other groups had shown that 

phytoplankton was diminished by atrazine by 50-200 μg/L although recovery of 

phytoplankton was common. 

It appears that most of the statistics presented did not look at the individual effects of 

atrazine exposure, that is, in the absence of carbaryl exposure. Therefore, the atrazine 

exposure results as presented in the article may be confounded by inclusion in the 

analysis of treatments containing atrazine and carbaryl and ‘control ponds’ without 

atrazine but containing carbaryl. There was little raw data presented to tease out the 

individual effects of atrazine, therefore the results should be interpreted with caution. 

Background concentrations of the pesticides in neither the source water nor the controls 

were reported. 

The treatment was limited to one test concentration of atrazine (200 ug atrazine/L). 

Atrazine was applied as part of a formulated product (Aatrex). It is unknown if there 

were any effects due to other ingredients in the formulation. 

There was an acclimation period but its duration is unclear. Natural phytoplankton 

communities appear to have been added to each mesocosm twice. An initial 500 ml of 

plankton was used to establish the ponds and acclimate. Then, the pesticides were mixed 

with 5 L of pond water of unknown source and added to the mesocosms. It is unclear if 

the pond water used for adding the chemical treatment had phytoplankton in it and if so 

how much. Based on the mean chlorophyll concentrations presented in Figure 1, 

chlorophyll concentrations appear similar prior to and a few days after application. It 

would be helpful to know more about the methodology to assess if the results may have 

been confounded by addition of more phytoplankton with pesticide application. 

Description of Use in Document: Qualitative

Rationale for Use: There was one endpoint relevant to aquatic plants measured in this 

study (chlorophyll content). There was an observation of reduced chlorophyll relative to 

controls at Day 12; however, the reduction was somewhat slight and transient (effect 

observed at Day 12, but not at the next measurement on Day 25). In addition, there was 

no indication of statistical significance at Day 12 (i.e., if atrazine-only treatments were 

statistically analyzed separately) and there was no indication of variability. Chlorophyll 

content was higher than controls at Day 25. Therefore, this observation was not 

considered to be treatment related. 
56

Limitations of Study: Results have good applicability to mesocosms or natural aquatic 

systems; however, the focus of the study was the effects of atrazine and carbaryl on the 

amphibian community and not the effects on phytoplankton. Also, the study design, the 

analysis, and the presentation and discussion of results of the study focused on interaction 

of factors, as opposed to the single effect of atrazine. No statistics or information about 

variation among replicates was provided regarding atrazine-only treatments. In addition, 

lake water was used, and there was no indication of whether or not atrazine may have 

been present in controls. 

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc 

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

Tertiary Reviewer: Brian Anderson, Biologist, EPA 

 
57

3.6. Brodeur, J.C., G. Svartz, C.S. Perez-Coll, D.J.G. Marino, J. Herkovits 2009. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: E112588; Brodeur, J.C., G. Svartz, C.S. 

Perez-Coll, D.J.G. Marino, J. Herkovits 2009. Comparative susceptibility to atrazine of 

three developmental stages of Rhinella arenarum and influence on metamorphosis: Nonmonotonous acceleration of the time to climax and delayed tail resorption. Aquatic 

Toxicology. 91 (161-170). 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Registration Review to evaluate effects to amphibians 

Date of Review: 11/21/11 

Summary of Study Findings: 

The objective of the study was to evaluate the acute and chronic toxicity of atrazine in R. 

arenarum embryos (stage 4), premetamorphosis (stage 25) and prometamorphosis (stage 

38-39). 

 Methods 

Adult South American toads (Rhinella arenarum) were obtained from a local provider. 

Ovulation induced by injecting homologous hypophysis and oocytes fertilized in vitro 

using fresh sperm. Embryos maintained until reaching the required stage for testing. 

Larvae were fed boiled swiss chard when reached stage 24-25. 

Technical grade atrazine was used (98%). 0.2% acetone was added to test solution to aid 

in dissolution to the 30 mg/L group. AMPHITOX solution (AS) used as dilution water 

solution. 

Studies conducted using stage 4 embryos (jelly coat removed prior to exposure) and 

Gosner stage 25 (premetamorphosis larvae) were conducted by placing 10 eggs or larvae 

into each of three replicates glass Petri dishes (containing 40 mL of test solution) and 

monitoring them for survival and malformations for 21 days. Nominal concentrations 

were negative and solvent control, 1.0, 2.5, 5, 7.5, 10, 15, 20, 25, and 30 mg/Land the 

solutions were replaced every 48 hours. Survival was monitored every other day. When 

larvae were at stage 25, a piece of boiled swiss chard was added. The study using 

embryos was repeated twice with a distinct pair of parents for each study. The study 
58

using premetamorphosis larvae was repeated four times with a distinct pair of parent each 

time. 

Another 4 studies using premetamorphosis larvae was conducted for 21 days at 

concentrations of 0.0001, 0.001, 0.01, 0.1 and 1.0 mg/L. 

A study which used larvae that had reached stage 38-39 (two completed back legs) were 

exposed to atrazine (0.1, 1, 5, 10, 15 and 20 mg/L) until completion of metamorphosis. 

Ten larvae per replicate (glass petri dishes w/ 100mL solution) with three replicates per 

treatment. Solutions were replaced every 48 hours and replicates maintained at 20 ±2°C. 

Swiss chard was placed in replicates as a food source. This study was conducted once 

with a single pair of parents. 

Results from the control and solvent control were compared using a t-test and as no 

difference was observed, the data was combined for further analysis. LC10, 50 and 90 

values were fitted using a four-parameter logistic regression equation. LC50 values for 

stage 25 larvae and larvae exposed at stage 4 were compared when the values for the 

stage 4 were adjusted to compare to stage 25 exposure. Percentages of malformations in 

embryos were calculated using ANOVA. For the prometamorphosis larvae the time for 

50% of larvae to reach stage 42 or complete metamorphosis were analyzed (ANOVA w/ 

Holm-Sidak test). The time intervals between stage 42 and completion was also 

analyzed. 

Results 

Measured concentrations (n=2) were <detection limit (0.02 mg/L), 1.3, 2.2, 3.9, 6.0, 10.4, 

13.8, 18.9, 21.5, and 25.9 mg/L for the nominal concentrations of 0, 1, 2.5, 5, 7.5, 10, 15, 

20, 25 and 30 mg/L, respectively. The reported LC values are presented below. For the 

stage 4 embryos, the authors reported that 10 days of exposure was needed to observe a 

significant decrease in survival whereas for larvae exposed at stage 25, significant 

decrease in survival occurred after 4 days of exposure. For the stage 4 embryos, the 

progression to stage 25 was similar among controls and treatments with concentrations of 

15 mg/L and higher slightly lagging behind. The incidence of malformations was not 

significant for any treatment group. 

Days of exposure Exposure from stage 4 

LC10 (mg/L) LC50 (mg/L) LC90 (mg/L) 

10 15.31 (11.64–20.14) 25.37 (22.86–28.15) N.A. 

14 7.84 (6.02–10.21) 14.41 (12.79–16.23) 26.42 (20.99–33.34) 

21 3.51 (2.56–4.81) 7.15 (6.31–8.09) 14.55 (11.12–19.05) 

Days of exposure Exposure from stage 25 

LC10 (mg/L) LC50 (mg/L) LC90 (mg/L) 

2 25.76 (22.08–30.13) N.A. N.A. 

4 18.28 (16.18–20.65) 27.16 (26.01–28.35) N.A. 

7 13.61 (11.86–13.63) 20.11 (18.99–21.3) 29.72 (26.30–33.57) 

10 8.04 (6.48–9.98) 14.48 (13.14–15.97) 26.06 (21.58–31.48) 

14 3.36 (2.75–4.12) 7.03 (6.48–7.62) 14.69 (12.36–17.46) 

21 0.71 (0.31–1.61) 2.32 (1.71–3.17) 7.59 (4.85–11.9) 

Days of exposure Exposure from stage 38–39 

LC10 (mg/L) LC50 (mg/L) LC90 (mg/L) 

7 16.26 (15.0–17.66) N.A. N.A. 

14 12.65 (11.17–14.32) 18.27 (17.38–19.20) N.A. 
59

Progression through Gosner stages until reaching stage 25 of embryos of R. arenarum exposed to atrazine from stage 4 

Days C At1 At2.5 At5 At7.5 At10 At15 At20 At25 At30 

0 4 4 4 4 4 4 4 4 4 4 

1 12 12 12 12 12 12 12 12 12 12 

2 17 17 17 17 17 17 17 17 17 17 

3 18 18 18 18 18 18 18 18- 18- 18- 

4 20 20 20 20 20 20- 20- 20- 19 19 

5 23 23 23 23 23 23 23- 23- 23- 23- 

6 24 25 25 24 24 24 24 23 23 23 

7 25 25 25 25 25 25 24 24 24 24 

8 25 25 25 25 25 25 25 25 25 25 

The number of the Gosner stage reached by the embryos after a given number of days of exposure are indicated. The number at the side of the 

abbreviation “At” represents the concentration of atrazine in mg/L. Controls are denoted by C. As all individuals of a given treatment normally evolved 

through Gosner stages at the same time, standard errors could not be calculated. A minus sign next to a Gosner stage number indicates embryos 

presenting an early version of the given stage.

In the study using stage 25 larvae and low levels of atrazine, the authors report that none 

of the concentrations below 1 mg/L caused significant decrease in survival (Figure 

below). 

Fig. 2. Concentration–response curves describing the survival of larvae of R. arenarum when exposed from stage 25 to low concentrations of atrazine 

for 7, 14 and 21 days. * = significantly different from controls (p < 0.05).

The study author reports that as embryos developed into stage 25 after 7-8 days with 

significant decrease in survival at Day 10 is similar to the mortality observed in larvae 

exposed at stage 25 when mortality was observed 2-3 days after exposure. For the larvae 

exposed at stage 38-39, no larvae survived at 15 and 20 mg/L with less than 50% survival 

at 10 mg/L. The reviewer assessed control mortality by using the Figures in the study, 

and it appears that control survival was 80% by Day 21 in the stage 4 studies and 90% in 

the stage 25 studies, and was 95-100% for the stage 38-39 studies. 

21 11.51 (10.45–12.71) 14.43 (14.05–14.83) 18.07 (16.87–19.41) 

28 10.02 (9.0–11.14) 12.63 (11.89–13.42) 15.92 (14.72–17.26) 
60

The study author states that of the three stages tested, stage 25 was the most sensitive 

stage, however, once embryos exposed at stage 4 reached stage 25, lethality was greater 

over next 10 days as stage 25 compared to lethality observed in larvae first exposed at 

stage 25. Figures and text show that none of the larvae in the 15 and 20 mg/L groups 

reached stage 42 or complete metamorphosis and only 20% at 10 mg/L. The authors 

state that time to reach stage 42 was accelerated by atrazine with a U shaped curve with 

0.1 and 1 mg/L having faster times and 5 mg/L having no difference compared to control. 

However, the time to complete metamorphosis showed no difference for 0.1 mg/L, 

decrease for 1 mg/L and increase for 5 mg/L. They state that this can be understood by 

analyzing the interval between stage 42 and completion as 0.1 and 5 mg/L are greater 

than control and 1 mg/L; however, the atrazine groups are not significantly different from 

one another. 

Fig. 6. (a) Time required (mean±S.E.) for 50% of the larvae of R. arenarum to reach stage 42 and completemetamorphosis when exposed to atrazine. 

(b) Duration of the interval (mean±S.E.) between when 50% of R. arenarum larvae reach stage 42 and when 50% complete metamorphosis when 

exposed to atrazine. At 0.1 = 0.1 mg/L of atrazine, At1=1mg/L of atrazine, and At5=5mg/L of atrazine. Controls are denoted by C. Bars with the same 

letter are not significantly different (p < 0.05). 

The authors state that while the bar graphs indicate that the difference between the 

control and treatment group 1 and 5 mg/L is 2 days, if compare to cumulative graph for 

completion, the difference translate to a 4 and 6 day difference when discussing time 

needed for 80-90% metamorphosis. Given that temporary ponds typical of R. arenarum

habitat, this difference may impact the larvae but research into features at metamorphosis 

and physiological integrity need to be examined. They also state that this study suggests 

that it is unlikely for atrazine to cause direct mortality to R. arenarum under typical 

conditions for use. They state that the difference in low level toxicity in this study 
61

compared to others need to be examined to determine if it is due to specific attributes of 

R. arenarum or a component of the formulations used in the other studies. 

Description of Use in Document (QUAL, QUAN, INV): 

Qualitative use for direct mortality (LC50 values), but invalid for use in comparisons for 

completion of metamorphosis. 

Rationale for Use: 

The study provides LC50 values for a toad species (although not native to U.S.) that can 

provide insight into acute and chronic mortality of amphibians. This taxa is not 

represented in routine testing of pesticides and represents an under representative taxa. 

Limitations of Study: 

The report does not indicate if the parental toads might have been previously exposed to 

potential contaminants as they only indicate they were from a local provider. Also, the 

report does not indicate or report levels of potential contaminants for the swiss chard food 

and water source. Loading rates were not reported nor were environmental conditions 

except for temperature. 

The concentrations in the low dose survival studies using the stage 25 larvae appear to 

not have been analyzed presumably due to being below the detection limit. 

For the embryo studies, the embryos were de-jellied prior to exposure. The reviewer 

assumes this was conducted for ease of distribution and potentially more uniform 

exposure. However, this process of removing the jelly may not reflect exposure 

conditions in the environment and make the embryos more susceptible to exposure 

uptake. 

Acetone was added to the 30 mg/L group, but a solvent control for this study does not 

appear to have been concurrently conducted. 

The non-monotoic dose-response observed in the metamorphosis study makes the ability 

to make a definitive conclusion about the effects of atrazine on metamorphosis more 

difficult. 

The author states that the bar graphs show only a 2 day difference between the 1 and 5 

mg/L groups, but the cumulative graphs indicate that the difference is 4 or 6 days. As the 

larvae were observed daily [reviewer assumes once daily], 1 day is the level of 

refinement for this measurement. Also, based on Figure 6a, it appears that the difference 

between the control and 5 mg/L is about 1 day not 2. 

For the studies, a single pair of parents was used for each experiment. For the embryos 

and stage 25 larvae, the experiment was repeated twice or four times, respectively. 

However, using organisms from a single parental source limits the genetic variability 

within a study which may confound results. Furthermore, the study which evaluated 

complete metamorphosis was only conducted once with a single pair of parents and this 

limits the ability to interpret effects of atrazine on larvae. However, the use of a single 

clutch may provide more consistent results within the study. 

The results of the metamorphosis endpoint were reported using EC50 values. It is not 

known why the results were analyzed using EC50 values instead of the results at test 

termination. The authors conduct the analysis of time to stage 42 and complete 
62

metamorphosis for 50% of the organisms but then discuss the environmental relevance in 

context of a 80 to 90% metamorphosis without conducting statistical analysis. The 

reasoning for conducting the analysis on when 50% of the organisms are developed as 

oppose to say when all are developed or test termination is not discussed. 

The authors state that atrazine significantly accelerated metamorphosis and had a nonmonotonic dose-response (U shaped) curve; however, given that this study was only 

conducted once using eggs from a single pair of toads, there is uncertainty in 

extrapolating these results to a general statement. In addition, while the time for 50% of 

the larvae to reach stage 42 and/or complete metamorphosis was significantly different 

from controls for one or more of the treatment groups not affected by survival, there was 

no significant difference between the treatment groups for the duration between when 

50% reached stage 42 and complete metamorphosis. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
63

3.7. Brodkin, M.A., H. Madhoun, M. Rameswaran, and I. Vatnick. 2007. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Brodkin, M.A., H. Madhoun, M. 

Rameswaran, and I. Vatnick. 2007. Atrazine is an immune disruptor in adult northern 

leopard frogs (Rana pipiens). Environ. Toxicol. Chem. 26 (1):80-84. ECOTOX 103351 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to examine if atrazine was an immune disruptor in Rana 

pipiens by measuring activity of white blood cells as an indicator of innate immune 

response. 

Methods 

Two experiments were conducted in which adult Rana pipiens of both sexes were used; 

frogs purchased from Amphibians of North America (Nashville, TN) and were field 

collected and prior exposure is not known. In the first experiment, twenty-four frogs 

were exposed to four different treatments (n=6/treatment): a 21 μg/L atrazine group 

injected with Ringer solution (RA), a 21 μg/L atrazine concentration followed by 

thioglycollate stimulation, an inflammatory mediator, (TA), a group only exposed to TN, 

and a group that was only injected with RN with the last two not containing atrazine. In 

the second experiment, 36 frogs were allocated into six treatments (n=6/treatment): 

atrazine concentrations of 0.01, 0.1, 1.0 and 10 μg/L which all received thioglycollate 

stimulation (TA), one control group that only received injection with Ringer solution 

(RN) and one group that only received thioglycollate stimulation (TN). The frogs were 

maintained individually in Tupperware containers with 500mL solution at 25°C. Frogs 

were injected on seventh day with Ringer solution or thioglycollate and euthanized on 

day 8 and a peritoneal lavage was performed with Ringer solution. Blood and livers were 

collected. Phagocytic activity was assessed using titrated beads with 100 or more white 

blood cells counted for each frog. Data from first experiment was expressed at total % 

phagocytic cells and from number 2, as either nonphagocytic or highly phagocytic. 

Results 

In experiment #1, the authors state that atrazine at 21 μg/L significantly diminished 

innate immune response when compared to frogs not exposed to atrazine but injected 

with thioglycollate (an inflammatory mediator) as the number of peritoneal exudates cells 
64

was significantly decreased. In experiment #2, all atrazine treatments has significantly 

decreased number of peritoneal exudates cells compared to thioglycollate control (see 

figures below). A decrease in the number of highly phagocytic cells was significant less 

at 0.01-10 compared to the thioglycollate stimulated control and 10 μg/L group was not 

significant compared to the Ringer solution group but the other groups were. 

 

Description of Use in Document (QUAL, QUAN, INV):

INVALID 

Rationale for Use: While the study included controls to that incorporated similar 

techniques as were used in the atrazine groups, a control group that was not exposed to 

atrazine or receive any other treatment was not included. 
65

Limitations of Study: A blank control was not used in the study in an effort to monitor 

adult performance of the population. This is to evaluate the activity of the frogs in the 

absence of any other treatment even if further comparison to the treatment groups may 

not be wholly adequate. While the study reported a change in white blood cells, these 

endpoints were not clearly and quantiatively linked to effects on survival, growth or 

reproduction in this study. Frogs were cultured using Styrofoam and maintained in 

plastic containers during the test and these materials are not recommended test materials. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
66

3.8. Choung, C.B, R.V. Hyne, R.M. Mann, M.M. Stevens, G.C. Hose. 2011. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Choung, C.B, R.V. Hyne, R.M. Mann, M.M. 

Stevens, G.C. Hose. 2011. Developmental toxicity of two common corn pesticides to 

the endangered southern bell frog (Litoria raniformis). Environ. Pollul. 159: 2648-2655. 

(ECOTOX Ref No. 153858) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/3/12 

Summary of Study Findings: 

The objective of the study was to examine the effects, singularly and in combination, of 

terbufos sulfone and atrazine on southern bell frog (SBF) larval development, growth and 

metamorphosis. This review will focus on the tests conducted with atrazine. 

Methods 

A pilot study was conducted in which the study authors stated that there was no 

significant difference in development rate between control and a 25 μg/L atrazine (>98%) 

treatment; however, the control tadpoles developed more quickly than the treatment with 

the combined pesticides. For the definitive test, gosner stage 26 tadpoles (ca. 6 weeks 

post hatch; obtained from laboratory-reared spawns) were exposed to a 25 μg/L atrazine 

concentration along with a blank (filtered, aged Sydney Australia tap water) control and a 

mixture of 25 and 10 μg/L atrazine and terbufos sulfone, respectively, for 10 weeks to 

encompass complete metamorphosis completely. There were three replicates (glass 

aquariums) for each treatment with 10 tadpoles in each replicate (10L solutions); test 

vessels aerated and maintained at 25oC under a 12:12 light:dark period. Test solutions 

renewed twice weekly along with measured test concentrations (duplicate samples) and 

environmental quality (dissolved oxygen, electrical conductivity, pH, and temperature). 

Each week each tadpole was photographed to examine visual inspection of physical 

abnormalities and measure snout-vent length (SVL) and tadpole stage. When tadpoles 

reached stage 42, they were transferred to another test vessel containing small amount of 

test solution until tail resorpbtion was complete at which time SVL and weight were 

measured. 

Results 

The results presented will focus on the definitive test. There were three mortalities 

overall, one in terbufos and two in combined, with all other tadpoles completing 
67

metamorphosis. There was no significant difference in survival, SVL, mass, or 

metamorphosis between the control and atrazine-only group (Figure 3). There was a 

significant delay in development in the combined group as well as the terbufos only 

group. The study authors state that atrazine did not interact synergistically with terbufos 

sulfone or on growth and development; although there was some indication of accelerated 

metamorphosis in the pilot study this should be interpreted with caution as the definitive 

test no differences were observed [results not shown]. 

Based on Figure 1B, it appears that the mass of the frogs at completion of metamorphosis 

in the control was ca. 0.87 – 1.01 grams with the mass in the atrazine only group around 

1.00 grams (Figure 1). As there was 10 tadpoles/10L water, this would be about 1 g/L at 

the time of metamorphosis. 

 

Fig. 1. (A) Mean snoutevent length of developing Litoria raniformis tadpoles and (B)final mass of emerged 

metamorphs exposed to atrazine, terbufos sulfone (T. sulfone) and the mixture of atrazine and terbufos 

sulfone (Mix) compared to the water-only control. Error bars in A represent standard errors. Dashed and 

solid lines within each box-plot in B represent the mean and median respectively.

Fig. 3. (A) Cumulative percent 

metamorphosis of Litoria raniformis 

tadpoles and (B) the average number of days 

taken by tadpoles to complete 

metamorphosis in the 
68

water-only control, atrazine, terbufos sulfone 

(T. sulfone) and the mixture of atrazine and 

terbufos sulfone (Mix) compared to the wateronly control. Treatments that were significantly 

different from control are indicated by * (p < 

0.05) and * * (p < 0.01). Dashed and solid lines 

within each box-plot in B represent the mean 

and median respectively.

Description of Use in Document 

(QUAL, QUAN, INV): QUAL 

Rationale for Use: 

While this study provides some 

evidence of the effects of atrazine on 

SBF at 25 μg/L, the lack of multiple 

doses precludes the ability to determine 

a dose-response. 

Limitations of Study: 

Statements about analysis of dilution 

water (dechlorinated tap water) and 

feed for potential contaminants not 

provided. 

Number of pairs of adults used to collect eggs for the definitive test is not provided; 

speaks to the potential genetic diversity in the test. 

The proportion data failed assumptions and was analyzed with a 0.01 α level. The reason 

for reducing the α level instead of using another statistical test is not known. It is also not 

known what effect lowering the α level had on the results. 

Given that there was only one atrazine concentration tested in this study, an evaluation of 

dose-response cannot be determined. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
69

3.9. Clements, C., S. Ralph, and M. Petras. 1997. 

OPEN LITERATURE REVIEW

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 20274 

Clements, C., S. Ralph, and M. Petras. 1997. Genotoxicity of select herbicides in Rana 

catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis 

(comet) assay. Environ. Mol. Muta. 29:277-288. 

Purpose of Review: Registration Review

Date of Assessment: 03/05/12 

Brief Summary of Study Findings: 

The objective of the study was to examine whether herbicides can increase DNA damage 

in Rana catesbeiana tadpoles. The study examined effects from 5 herbicides but only 

results of atrazine presented. 

R. catesbeiana tadpoles were received from Carolina Biological Supply Company 

(CBSC); cultured in polypropylene containers containing dechlorinated water under static 

renewal conditions (48 hr renewal) and fed tadpole food from CBSC and maintained for 

at least 1 week prior to testing. Tadpoles were exposed in the dark to formulations 

(AAtrex Nine-O (atrazine); Dual-960E (metalochlor), Roundup (glyphosate), Sencor500F (metribuzin), and Amsol (2,4-D amine)), methy methanesulfonate (MMS) or 

dechlorinated water in for 24 hours in 1L beakers containing 500 mL test solution. Test 

concentrations for the herbicides included the maximum survival concentrations and 1⁄4 

and 1/6 dilutions of this concentration; MMS concentration was 3.125 mg/L. After 24 

hours, blood samples collected (using Hank’s balanced salt solution after decapitation) 

from tadpoles with erythrocytes being the primary cell collected. Slides prepared and 

examined and length and width of DNA mass determined. 

All AAtrex Nine-O concentrations (4.81, 19.25, and 77 mg/L) had significant increases 

(p<0.001) in DNA ratios relative to control (Table 1). All animals died within 24 hours 

at 308 mg/L [272 mg a.i./L-reviewer calculated]. The linear correlation between DNA 

damage and AAtrex concentration was moderate according to the study authors (r=0.663, 

P<0.001). 
70

 

Description of Use in Document: Invalid. 

Rationale for Use: Limited details on study design including number of organisms 

exposed and number of replicates. 

Limitations of the Study: Limited details on study design (number of organisms 

exposed, number of replicates); author states procedure described in Ralph et al 1996 

however, it appears to be for DNA assay not test design [Ralph S., Petras M., Pandrangi, 

R., Vrzoc, M. 1996. Alkaline single cell gel (comet) assay and genotoxicity monitoring 

using two species of tadpoles. Environ. Mol Mutagen. 28:112-120.]. Mortality not 

reported and with different number of animals used in the assay for each concentration 

and no details on test design, the reviewer cannot discern mortality results. Water quality 

parameters not reported. Percent active ingredient not reported, reviewer assumed based 

on available label (88.2% a.i.). As the exposure was to a formulation, it is unknown if the 

observed toxicity was due to technical atrazine, an ingredient(s) in the formulation or 

some combination. Based on the concentrations tested, the two highest concentrations 

tested appeared to be above the water solubility of atrazine (ca. 33 mg/L). While it 

provides some information on DNA damage, the results are not directly correlated to 

survival, growth or reproduction in this study. 

Reviewer: Amy Blankinship, Chemist, Environment Risk Branch 3, Environmental Fate 

and Effect Division. 

 
71

3.10. Diana, S. G., Resetarits, W. J. Jr., Schaeffer, D. J., Beckmen, K. B., 

and Beasley, V. R. (2000). 

Chemical Name: Atrazine (Amphibian Microcosm Study) 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 59818 

Diana, S. G., Resetarits, W. J. Jr., Schaeffer, D. J., Beckmen, K. B., and Beasley, V. R. 

(2000). Effects of Atrazine on Amphibian Growth and Survival in Artificial 

Aquatic Communities. Environ.Toxicol.Chem. 19: 2961-2967. 

Purpose of Review: Litigation

Date of Assessment: 3/20/06 

Brief Summary of Study Findings: 

The goal of the study was to determine the effects of atrazine on survival, mass, and 

length at metamorphosis, and days to metamorphosis of larval gray tree frogs (Hyla 

versicolor) inhabiting artificial pond microcosms and to assess interrelationships among 

these parameters and DO concentrations, water pH, and estimates of phytoplankton, 

periphyton, and macrophyte biomass. 

Artifical pond microcosms (16 plastic wading pools; 1.22-m diameter; arranged in 4x4 

array on gravel pad) with 90 L pond water (includng phytoplankton, macrophytes) were 

used. 40 larvae were added to each microcosm and treated with technical grade atrazine 

to achieve final aqueous concentrations of 0, 20, 200 or 2000 ppb (4 reps of each 

treatment and control; acetone was used as solvent in treatment groups and solvent 

control). Microcosms were covered with mesh fiberglass to exclude predators. 

Plants: Phytoplankton chlorophyll a concentrations declined slightly during the 1st week 

following atrazine addition (in all but 200 ppb group) and, by Day 14, rebounded above 

levels before exposure (in all but the 20 ppb group). Phytoplankton densities in the 200 

and 2000 ppb groups increased significantly above control during rebound period. Over 

course of study (~40 days), chlorophyll a conc. Was lowest in control, highest in 200 

ppb, and intermediate in 20 and 200 ppb groups. Macrophyte biomass at the end of the 

study was decreased, relative to controls, by 30%, 98%, and 99% in the 20, 200, and 

2000 ppb groups, respectively. 

Water chemistry: DO decreased to approx 20 and 40% of their pre-exposure values in 

the 200 and 2000 ppb treatment grps within 1 d of atrazine addition. DO in these 

microcosms returned to control concs by 10 d after exposure but declined again to approx 

60 to 80% of control values at 21 d after exposure and remained depressed for the 

remainder of the study. In the 200 and 2000 ppb grps, pH decreased similarly within 1 d 

of atrazine exposure and returned to control values within the following 16 d. The DO 
72

and pH did not differ significantly between the 0 and 20 ppb groups or the 200 and 2000 

ppb groups. 

Amphibians: Frogs from the two higher atrazine conc treatment groups were 5% shorter 

and had 10% lower body mass at metamorphosis than those from the control and low 

atrazine groups. No difference in length or body mass at metamorphosis was detectable 

between the 0 and 20 ppb groups or between the 200 and 2000 ppb groups. Larval period 

was 5% longer in the 2000 ppb groups than in the 200 ppb group, but did not differ from 

controls in any treatment group. No significant treatment-related differences were 

detected for survival rate. 

The decrease in amphibian length and weight at metamorphosis may indicate a 

reduction in fitness of wild populations of anurans exposed to atrazine at 200 to 

2000 ppb. The NOAEL is between 20 and 200 ppb. 

Given lack of decrease phytoplankton over time and subsequent compensatory growth of 

phytoplankton following exposure, it seems unlikely that the effects on amphib 

development were due to a decrease in food. However, atrazine-resistant species 

occurring in presence of continued atrazine exposure may be less palatable, of lower 

nutritive value or toxigenic. Rebound of phytoplankton likely due to elimination of 

macrophytes. Adverse effects of atrazine on amphibian growth are mediated primarily 

by decreased oxygen availability. Larvae impacted by reduced DO at 200 and 2000 ppb 

levels – in other species larvae increase effort at gill respiration, at the expense of 

feeding, when held in water with low DO. 

Based on observed decreases in length and mass at metamorphosis, and decreases in pH, 

DO, and macrophyte biomass, it is suggested that these variables may lead to increased 

risks of predation as well as decreased fitness.

Description of Use in Document: Qualitatively (based on plant cosm classification). 

Rationale for Use: Provides information on development and growth. 

Limitations of the Study: No raw data provided; pre-metamorphosis weight and length 

were not determined. 

Reviewer: Anita Pease, ERB3 

Addendum to Open Literature Review 

3/5/12 

Limitations of Study: 

Plastic wading pools were used as test vessels and therefore potential contaminants 

(leaching from pool) may have been present. 
73

Atrazine was measured immediately following its addition to the test vessels. The 

measured concentrations (mean ± SE) for the 0, 20, 200 and 2,000 μg/L groups were 1.64 

± 0.09, 20.13 ± 0.58, 195.50 ±8.74 and 2,035.83 ± 51.54 μg/L, respectively. Therefore, 

atrazine appears to have been measured in the control at about 8% of the lowest test 

concentration and 0.8% of the middle concentration which is the level where significant 

effects were observed. 

Reviewer: Amy Blankinship, Chemist, ERB3, OPP 

 
74

3.11. Du Preez, L. H., N. Kunene, G. J. Everson, J. A. Carr, J. P. Giesy, T. 

S. Gross, A.J. Hosmer, R. J. Kendall, E.E. Smith, K. R. Solomon, G.J. Van 

Der Kraak. 2008. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Du Preez, L. H., N. Kunene, G. J. Everson, 

J. A. Carr, J. P. Giesy, T. S. Gross, A.J. Hosmer, R. J. Kendall, E.E. Smith, K. R. 

Solomon, G.J. Van Der Kraak. 2008. Reproduction, larval growth, and reproduction 

development in African clawed frogs (Xenopus laevis) exposed to atrazine. 

Chemosphere 71:546-552. 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to evaluate effects over a multigeneration with breeding 

evaluated from frogs exposed to atrazine with effects on number of eggs released, 

hatching success, and development of F2 individuals monitored. 

This study is a continuation of the cosm study by Jooste et al. 2005 (46965603). 75 

Xenopus laevis metamorphs from each atrazine concentration (0, 1, 10, and 25) were 

placed into 2500L tanks in lab and exposed to same atrazine concentration. After cool 

months, frogs placed back into tanks outdoors. After 1 year frogs were placed into 30L 

glass tanks at 6 frogs/tank at same atrazine concentration. After two years grow out 

males and females were paired for breeding. Spawning was induced by injection. Clutch 

size, hatching success were evaluated and F2 tadpoles allowed to complete 

metamorphosis after which time they were processed and evaluated for gross pathology 

and histopathology. 

According to the authors the size of the females influenced the clutch size with large 

differences in number of eggs. Also, the authors state that there was no effect on clutch 

size, hatching success, growth or sexual development in the study. 

Description of Use in Document (QUAL, QUAN, INV):

INVALID 
75

Rationale for Use: 

This study was a continuation of another study (Jooste et al. 2005) and that study has 

uncertainties that deemed it INVALID. 

Limitations of Study: Those uncertainties in Jooste et al. (2005) were: Unusually long 

development times; feeding and temperatures may have limited development. Higher 

incidence of testicular oocytes in controls. In this study, hatching success in the controls 

varied greatly (36-96%). 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
76

3.12. Forson, D. and Storfer, A. (2006). 

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 82033 

Forson, D. and Storfer, A. (2006). Effects of Atrazine and Iridovirus Infection on 

Survival and Life-History Traits of the Long-Toed Salamander (Ambystoma 

macrodactylum). Environ.Toxicol.Chem. 25: 168-173. 

Purpose of Review: Registration Review

Date of Assessment: 5/1/12 (this review relies on the review done on 4/7/06) 

Brief Summary of Study Findings: 

Recent studies suggest that agricultural contaminants, such as atrazine, may have 

suppressive effects on the amphibian immune system, thereby increasing susceptibility to 

parasites and pathogens such as iridoviruses in the genus Ranavirus and the chytrid 

fungus (Batrachochytrium dendrobatidis). 

Study tested the interaction of emerging infectious diseases and atrazine (86.5% ai) in 

long-toed salamanders (Ambystoma macrodactylum). 6-week old long-toed salamanders 

were exposed to Ambystoma tigrinum virus (ATV; 0 or 103.5 plaque-forming units/ml) 

and sublethal concentrations of atrazine (0, 1.84, 18.4, and 184 ppb) in a 4x2 factorial 

design for 30 days. ATV is an emerging iridovirus responsible for epizootics in tiger 

salamanders through out western North America. The effects of atrazine and virus was 

tested on mass and snout-vent length (SVL) at metamorphosis and larval period as well 

as on rates of mortality and viral infectivity. ATV transmission was confirmed, although 

infection rates were lower than expected, consistent with the theory predicting lower 

pathogen transmission to nonnative hosts. Larvae exposed to both atrazine and ATV had 

lower levels of mortality and ATV infectivity (13.3% across all 3 atrazine concentrations) 

compared to larvae exposed to virus alone (25%), suggesting atrazine may compromise 

virus efficacy. The highest atrazine level (184 ppb) accelerated metamorphosis and 

reduced mass and SVL at metamorphosis relative to controls. Exposure to ATV also 

significantly reduced SVL at metamorphosis. Atrazine alone had no significant effect on 

mortality. 

The study suggests moderate concentrations of atrazine may ameliorate ATV effects on 

long-toed salamanders, whereas higher concentrations initiate metamorphosis at a smaller 

size, with potential negative consequences to fitness. Larger size at metamorphosis is 

correlated with higher survival to maturity and reduced time to maturity, thereby 

increasing fitness relative to smaller individuals [34-36 in paper]. Smaller size at 

metamorphosis may be a fitness cost resulting from high-level atrazine exposure. 

Lighter, smaller animals have reduced terrestrial locomotor performance and, therefore, 
77

reduced ability to avoid predators or capture prey [35,36]. Smaller, newly 

metamorphosed adults also tend to have weakened immune systems, which could make 

them more susceptible to disease. 

Methods:

200 eggs collected from a high altitude pond near St. Paul peak in Cabinet Mountain 

Wilderness Area (MT). Larvae fed 0.015 g brine shrimp 3x/wk for 6 wk, then 

blackworms 2x/wk ad libitum at 6 wks of age and throughout the experiment. Eight 

treatement groups (20 larvae/treatment for total of 160 individuals). Water was changed 

every 3 days. 

Description of Use in Document: Qualitatively 

Rationale for Use: Provided information on interaction of atrazine and virus on 

amphibian development. 

Limitations of the Study: No raw data provided, and not all summary data for different 

treatment groups provided (groups with ATV and AZT). Water quality parameters, other 

than temperature, were not reported. Reviewer assumed loading to be 1 larvae/500mL 

water which is lower than 1 tadpole or larvae/1L recommended by ASTM. Limited 

water quality data reported. 

Reviewer: Anita Pease, ERB3; Amy Blankinship, ERB3 

 
78

3.13. Forson, D. and Storfer, A. (2006). 

Chemical Name: Atrazine (Long-toed Salamander Study; effects of atrazine and 

iridovirus) 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 82033 

Forson, D. and Storfer, A. (2006). Effects of Atrazine and Iridovirus Infection on 

Survival and Life-History Traits of the Long-Toed Salamander (Ambystoma 

macrodactylum). Environ.Toxicol.Chem. 25: 168-173. 

Purpose of Review: Registration Review

Date of Assessment: 5/1/12 (this review relies on the review done on 4/7/06) 

Brief Summary of Study Findings: 

Recent studies suggest that agricultural contaminants, such as atrazine, may have 

suppressive effects on the amphibian immune system, thereby increasing susceptibility to 

parasites and pathogens such as iridoviruses in the genus Ranavirus and the chytrid 

fungus (Batrachochytrium dendrobatidis). 

Study tested the interaction of emerging infectious diseases and atrazine (86.5% ai) in 

long-toed salamanders (Ambystoma macrodactylum). 6-week old long-toed salamanders 

were exposed to Ambystoma tigrinum virus (ATV; 0 or 103.5 plaque-forming units/ml) 

and sublethal concentrations of atrazine (0, 1.84, 18.4, and 184 ppb) in a 4x2 factorial 

design for 30 days. ATV is an emerging iridovirus responsible for epizootics in tiger 

salamanders through out western North America. The effects of atrazine and virus was 

tested on mass and snout-vent length (SVL) at metamorphosis and larval period as well 

as on rates of mortality and viral infectivity. ATV transmission was confirmed, although 

infection rates were lower than expected, consistent with the theory predicting lower 

pathogen transmission to nonnative hosts. Larvae exposed to both atrazine and ATV had 

lower levels of mortality and ATV infectivity (13.3% across all 3 atrazine concentrations) 

compared to larvae exposed to virus alone (25%), suggesting atrazine may compromise 

virus efficacy. The highest atrazine level (184 ppb) accelerated metamorphosis and 

reduced mass and SVL at metamorphosis relative to controls. Exposure to ATV also 

significantly reduced SVL at metamorphosis. Atrazine alone had no significant effect on 

mortality. 

The study suggests moderate concentrations of atrazine may ameliorate ATV effects on 

long-toed salamanders, whereas higher concentrations initiate metamorphosis at a smaller 

size, with potential negative consequences to fitness. Larger size at metamorphosis is 

correlated with higher survival to maturity and reduced time to maturity, thereby 

increasing fitness relative to smaller individuals [34-36 in paper]. Smaller size at 

metamorphosis may be a fitness cost resulting from high-level atrazine exposure. 
79

Lighter, smaller animals have reduced terrestrial locomotor performance and, therefore, 

reduced ability to avoid predators or capture prey [35,36]. Smaller, newly 

metamorphosed adults also tend to have weakened immune systems, which could make 

them more susceptible to disease. 

Methods:

200 eggs collected from a high altitude pond near St. Paul peak in Cabinet Mountain 

Wilderness Area (MT). Larvae fed 0.015 g brine shrimp 3x/wk for 6 wk, then 

blackworms 2x/wk ad libitum at 6 wks of age and throughout the experiment. Eight 

treatement groups (20 larvae/treatment for total of 160 individuals). Water was changed 

every 3 days. 

Description of Use in Document: Qualitatively 

Rationale for Use: Provided information on interaction of atrazine and virus on 

amphibian development. 

Limitations of the Study: No raw data provided, and not all summary data for different 

treatment groups provided (groups with ATV and AZT). Water quality parameters, other 

than temperature, were not reported. Reviewer assumed loading to be 1 larvae/500mL 

water which is lower than 1 tadpole or larvae/1L recommended from 2007 SAP. Limited 

water quality data reported. 

Reviewer: Amy Blankinship, ERB3 

 
80

3.14. Fort, D. J., R. L. Rogers, J.H. Thomas, B.O. Buzzard, A. M. Noll and 

C.D. Spaulding. 2004 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 81751 

Fort, D. J., R. L. Rogers, J.H. Thomas, B.O. Buzzard, A. M. Noll and C.D. Spaulding. 

2004. Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species 

for the FETAX model. J. App. Toxicol. 24: 443-457 

Purpose of Review: Registration Review 

 

Date of Assessment: 5/1/12 

Brief Summary of Study Findings: 

Studies were conducted according to ASTM E1439-98 FEXTAX assay in regards to 

animal husbandry, breeding and embryo collection and testing for Xenopus laevis and X. 

tropicalis. Several compounds were tested but only atrazine will be review here. The 

studies were terminated at NF stage 46. 

The acute LC50 values for X. laevis and X. tropicalis range from 24.5-25.6 mg/L for 

LC50 and 19.4-27.5 mg/L, respectively. The acute EC50 values for X. laevis and X. 

tropicalis range from 4.1-4.4 mg/L and 2.1-9.3 mg/L, respectively 

 

Description of Use in Document: INVALID 

 

Rationale for Use: The study provides some information on the acute effects of atrazine 

on amphibian development, however, the test conditions in the FETAX assay tend not to 

be optimal for use in a risk assessment especially in solvent choice and concentration. 

Limitations of the Study: Water quality parameters not reported. DMSO was used at 

1% and DMSO is not a recommended solvent the recommended solvent concentration 

for acute assays is 0.05%. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
81

3.15. Freeman J.J. and A.L. Rayburn. 2004 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 78644 

Freeman J.J. and A.L. Rayburn. 2004. In vivo genotoxicity of atrazine to anuran larvae. 

Mut. Res. 560: 69-78. 

Purpose of Review: Registration Review 

 

Date of Assessment: 4/20/12 

Brief Summary of Study Findings: 

American toads (Bufo americanus) and Xenopus laevis tadpoles were exposed to atrazine 

and DNA from tadpoles was examined. B. americanus tadpoles were obtained from 

adults that were field collected and overwintered in lab; X. laevis tadpoles purchased 

from NASCO. In the first experiment for B. americanus, tadpoles (3-L tanks with 2 

tadpoles/tank) were exposed to atrazine concentrations of 0, 250, 500, 1000, 5000 and 

10000 ppb for three weeks. Both tadpoles processed for DNA analysis (nuclei). In 

second experiment, tadpoles (2/tank) were exposed to 0 and 800 ppb atrazine for three 

weeks and then processed for DNA analysis. For X. laevis, the first experiment consisted 

of 3-L tanks with 2 tadpoles/tank and tadpoles exposed to 0 and 800 ppb atrazine for 3 

weeks after which they were processed for DNA analysis. In the second experiment, X. 

laevis tadpoles were processed at 3, 4, and 5 weeks. Water was changed weekly and 

solutions analyzed for atrazine weekly; water conditions were monitored. 

Atrazine concentrations reported as stable between weekly renewals. According to the 

study authors, atrazine did not appear to be genotoxic for both species. 

Description of Use in Document: Qual: 

 

Rationale for Use: Provides information on genotoxic potential however, uncertainties 

in sample size and limited test design. 

Limitations of the Study: Water quality parameters not reported. Test solution 

preparation not reported, unsure if solvent used and what control represented. Test vessel 

material and loading not reported. Survival not reported. The number of replicates per 

treatment is not specified. The study authors state that the tank is the experimental unit 

with an ‘n’ of 6 for each treatment in the Figures which the reviewer assumes indicates a 

total of 6 replicates or 6 independent trials. Statistics completed using CVs instead of 

means for each treatment. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 
82

3.16. Freeman J.L. N. Beccue and A.L. Rayburn. 2005 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 81460 

Freeman J.L. N. Beccue and A.L. Rayburn. 2005. Differential metamorphosis alters the 

endocrine response in anuran larvae exposed to T3 and atrazine. Aqua. Tox. 75: 263-276. 

ECOTOX 81460 

Purpose of Review: Registration Review 

 

Date of Assessment: 5/1/12 

Brief Summary of Study Findings: 

American toads (Bufo americanus) were exposed to atrazine and DNA from tadpoles was 

examined. B. americanus tadpoles were obtained from adults that were field collected 

and overwintered in lab. In the first experiment for B. americanus, tadpoles (Gosner 25) 

(3-L tanks with 2 tadpoles/tank) were exposed to atrazine concentrations of 0 and 800 

ppb for three weeks. Both tadpoles staged, weighed and processed for DNA analysis 

(nuclei from whole body homogenates). This experiment was repeated 18 times. In 

second experiment, tadpoles (G20), obtained as eggs from pond in Alabama, (2/tank) 

were exposed to 0 and 800 ppb atrazine for four weeks of which they were staged, 

weighed and then processed for DNA analysis. This experiment was replicated four 

times. In the third experiment, tadpoles (G25, 2 per 3-L tank, obtained from adults 

collected in IL and overwintered) were exposed to 0, 250, 500, 1000, 5000 and 10000 

ppb atrazine for three weeks after which they were staged, weighed and processed. This 

experiment was replicated three times. There was one tank per treatment per test. Water 

was changed weekly and solutions analyzed for atrazine weekly; water conditions were 

monitored. 

In the first experiment, no acute toxicity observed and total number of nuclei were not 

significantly different; however, control tadpoles at significantly earlier stage and 

weighted less than tadpoles at 800 ppb atrazine (p=0.0007 and 0.0002). In the second 

experiment there were no effects on stage, weight or number of nuclei. For the third 

experiment no acute toxicity observed and after 3 weeks, no difference in total number of 

nuclei was observed and weight was not significantly different. However, tadpoles at 

atrazine concentrations of 250, 500 and 1000 ppb, but not 5000 and 10000 ppb, were at a 

significantly greater stage than then controls. 

Description of Use in Document: Qualitative for metamorphosis and nuclei analysis. 

Invalid for weight. 

 
83

Rationale for Use: While the study provides information on the development of B. 

americanus tadpoles, there are uncertainties in the loading and environmental conditions. 

In addition, there is uncertainty in the statistical outcome for weight given that tadpoles 

were at different stages when analyzed for weight. 

Limitations of the Study: Water quality parameters not reported. Test solution 

preparation not reported, unsure if solvent used and what control represented. Test vessel 

material and loading not reported. Survival not reported, however based on sample sizes 

provided it appears it was 100%. As tadpole weight changes and is linked with 

metamorphosis, analyzing weight for tadpoles that are at different stages confounds the 

results. It is unclear, if and how, the study authors accounted this in the study. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
84

3.17. Gibble, R.E. and K.N. Baer. 2011 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Gibble, R.E. and K.N. Baer. 2011. Effects of 

atrazine, agricultural runoff, and selected effluents on antimicrobial activity of skin 

peptides in Xenopus laevis. Ecotox. Environ. Saf. 74: 593-599. (ECOTOX Ref No. 

153856) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/27/12 

Summary of Study Findings: 

The objective of the study was to investigate the influence of common environmental 

pollutants on the production and in vitro bioactivity of protective peptides in an 

amphibian model (X. laevis). 

Methods 

The test materials used were technical grade and formulated atrazine (ATR) with 

surfactant, agricultural runoff containing a mixture of pesticides, fields treated with or 

seeds planted with a variety of chemicals: paraquat, captan, diazinon, lindane, smetolachlor, and atrazine. Also tested were treated sewage treatment plant and municipal 

landfill effluents. This review will focus on the technical grade and formulated atrazine. 

X. laevis embryos were purchased from Nasco as embryos and the chytrid fungus, 

Batrachochytrium dendrobatidis was purchased from University of Maine. NF stage 

49/50 larvae were exposed to atrazine concentrations of 0, 0 (DMSO, 0.0173% [reviewer 

calculated]), 0.1, 1, 10, 100 and 1000 μg/L under flow-through conditions with two 

replicates for each treatment group. Concentrations were measured weekly from 

alternate reps. Tadpoles (NF 49/50) were also exposed in a semi-static system (halfvolume renewals 3 times weekly) to the atrazine formulation AAtrex® 4L (40.8% ATR) 

mixed with Top Surf® surfactant at atrazine concentrations of 0.01, 1, 100 and 1000 μ 

a.i./L. The high concentration was verified prior to use and used to prepare remaining 

test solutions. Unfiltered water collected from Bayou Desiard was used as dilution water; 

this water is a drinking water source and water quality parameters are monitored monthly. 

Larvae were exposed till completion of metamorphosis (NF stage 66) or at test 

termination of 90 days or when 80% of the controls reach stage 66. Secretions were 

induced from groups of five post metamorphic animals and analyzed for protein content 

and bioactivity. Antimicrobial secretions were induced by subcutaneous injection of 
85

norepinephrine bitartrate into the right dorsal lymph sac of post metamorphic animals, 

secretions pooled from 5 animals, and peptide concentrations measured. B. dendrobatidis

zoospores were incubated with or without the peptides and read on Day 0 and 7 to 

evaluate cell growth and growth curves. 

Results 

Mean measured concentrations in the technical atrazine study were 79 to 105% of 

nominal, except for the 0.1 μg/L group which was 281% of nominal; atrazine not 

detected in control samples except for one sample in solvent control with measurement of 

0.16 ppb for up to potentially 48 hours. According to the study, in the agricultural runoff 

samples, atrazine concentrations were initially 1.0 μg/L but increased to a maximum of 

7.4 μg/L over ca. 8 weeks (1-2 weeks following second atrazine application). There were 

no effects on survival, growth or development (data not presented). There were also no 

differences in total protein or slope between technical atrazine concentrations and 

controls as well as no significant trend in total protein. For the atrazine formulation, 

ANOVA detected difference in total protein, but Tukey’s post-hoc test did not; however 

regression analysis indicated significant decreasing trend in total protein with increasing 

atrazine concentrations. Bioactivity was not significant between treatments for the 

atrazine formulation study. 

 

Description of Use in Document (QUAL, QUAN, INV): 

INVALD for growth, survival, metamorphosis endpoints due to lack of reporting results. 

QUAL for peptide analysis 

Rationale for Use: 

This study provides information on survival, growth and development, however, as no 

data were presented on these parameters, the reviewer cannot evaluate the study authors 

results therefore large uncertainty for these endpoints. In addition, for the technical grade 

atrazine study DMSO was used as the solvent which is not a preferred solvent as it 

promotes movement across membranes. 

Limitations of Study: 

The report did not specify what parameters in regards to survival, growth and 

development were analyzed. The study did not report results for these endpoints, 

therefore, the reviewer could not evaluate these parameters. Survival, to assess control 

performance, was not reported 

For the technical grade atrazine study, DMSO was used as the solvent at 0.0173% which 

is ca. twice the maximum limit for chronic testing under U.S. EPA OPPTS testing 

guidelines; in addition, DMSO is not a preferred solvent as it promotes movement across 

membranes. 
86

The study design in terms of replicates and number of organisms at each replicate for the 

AAtrex formulation study was not reported. The study did not report if the feed was free 

of potential contaminants. Atrazine was measured in the solvent control (0.16 ppb) in 

which the author states may have been present for up to 48 hours. Also, the test vessels 

were not described. 

The results for the peptide assays and water quality characteristics only report one value 

for the 0 μg/L concentration. Therefore, it appears that the results were pooled for the 

negative and solvent controls for the technical grade atrazine study, but this is not stated 

in the study. 

The reviewer was uncertain about the limits of the analytical method as the study reports 

that the limit of quantitation was 16 ng and the limit of detection was 0.1 ppb. 

Regardless, the lowest concentration in the technical grade and formulation atrazine study 

appear to be below the limit of analysis. In addition, the 0.1 ppb treatment group in the 

technical grade study was 281% of nominal and the report does not explain why this 

group is so high. 

For the peptide bioactivity, the study reports that groups of 5 frogs were pooled to obtain 

secretions. Based on the figures, the sample size is 6 suggesting that 30 frogs/treatment 

were used. There were a reported two replicates for the technical atrazine study with no 

test design reported for the formulation. Therefore, it appears that for this endpoint, 

groups of animals were treated as the experimental unit and not the tanks. The peptide 

bioactivity assay was conducted by inducing antimicrobial secretions in the animals with 

a subcutaneous injection, therefore there is uncertainty in the relevance of this exposure 

route in the environment. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 Not applicable 

 
87

3.18. Gunderson, M.P, N. Veldhoen, R.C. Skirrow, M.K. Macnab, W. Ding, 

G. van Aggelen, and C.C. Helbing. 2011 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Gunderson, M.P, N. Veldhoen, R.C. 

Skirrow, M.K. Macnab, W. Ding, G. van Aggelen, and C.C. Helbing. 2011. Effect of 

low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 

aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic 

bullfrog tadpoles (Rana catesbeiana). Aquat. Toxicol. 102: 31-38. (ECOTOX Ref. No. 

153613). 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/1/12 

Summary of Study Findings: 

The objective of the study was to examine whether atrazine and its degradate, 

desethylatrazine (DEA) alters the steady-state levels of SF-1 and CYP19 cDNA 

transcripts in the brain of premetamorphic bullfrog tadpoles. 

Methods 

Two experiments were conducted using Rana catesbeiana tadpoles (Talyor and Kollros 

stages VI-VIII obtained from Ward’s Natural Science (St. Catherines, ON, Canada), 

maintained in outdoor tanks at ca. 9°Cand acclimated indoors for ca. 48 hours at 22°C); 

tadpoles not fed 24 hours prior to experiment or during test. The studies were conducted 

exposing tadpoles to 673 ng/L T3 (thyroid hormone, 3,5,3’-triiodothyronine), 50 ng/L 

EE2 (17α-ethynylestradiol) and 1 and 100 μg/L DEA or atrazine for 48 hours; all 

treatments were prepared using isopropyl alcohol with a solvent concentration of 0.001% 

and a solvent control was included. Each treatment group had 5 tadpoles per replicate (20 

gal glass aquariums with 50L well water; ave. loading 0.63 g/L). Dissolved oxygen, 

temperature and pH recorded at test initiation and termination. Tadpoles lost weight 

during the 48 hour exposure period however it as not significant between treatments. 

After 48 hours, tadpoles were euthanized and whole brains isolated and stored. Total 

RNA was isolated from whole brain tissue and cDNA synthesis and development of 

species-specific QPCR primers and assays conducted. 

Results 

The isolated partial bullfrog CYP19 cDNA sequence showed >90% putative amino acid 

sequence similarity across four anuran species; ca. 89% similarity with two salamander 
88

species and 81% similarity with two 

fish species. The study authors 

indicate that there was a trend towards 

a decrease in CYP19 transcript levels 

in DEA groups (p=0.055 KrislaWallis) and a trend towards an 

increase for the atrazine exposed 

tadpoles (p=0.041- Kruskal-Wallis). 

No effect on SF-1 transcript was 

observed for atrazine or DEA 

treatment (Figure 1). A significant 

positive correlation between CYP19 

and SF-1 transcript abundance was 

observed in the atrazine treatment 

(correlation coefficient 0.526, 

p=0.017) but was not observed for 

DEA or T3 or EE2 groups (Table 1). 

The authors state that the results in the 

study support the hypothesis that 

atrazine induce feminization in the 

brain by increasing CYP19 mRNA 

levels with the results showing greater 

variation of CYP19 transcript in the 

atrazine groups than the solvent 

control. The authors state that support 

for atrazine induced feminization 

through the SF-1 transcript was not evident in this study as a correlation was observed in 

the atrazine group but not the EE2 group. 

Figure 1 (reprinted from Fig. 4.in the study) Relative fold difference in CYP19 and SF-1 mRNA 

abundance in whole brain tissue of premetamorphic R. catesbeiana tadpoles exposed for 48 h to isopropyl 

alcohol (IPA) vehicle alone or 1_g/l and 100_g/l of ATZ or DEA. A bevel indicates increasing 

concentrations of ATZ or DEA. The medians are shown as solid black lines within the box, and the box 

indicates the first and third quartiles. The whiskers indicate minimum and maximum values. Outlier (cases 

between 1.5 and 3.0 box lengths from the upper or lower edge of the box) and extreme values (cases >3.0 

box lengths from the upper or lower edge of the box) are indicated by an open circle and asterisk, 

respectively. 

Table 1 (reprinted from Table 3 in study) 
89

Description of Use in Document (QUAL, QUAN, INV):

INVALID 

Rationale for Use: 

Based on solvent choice and no negative control. 

Limitations of Study: 

The use of isopropyl alcohol is not a recommended solvent (ASTM). While the report 

indicates they were measured, results of the water quality parameters were not reported. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
90

3.19. Hayes, T. B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. 

Adame, E. Chan, D. Bucholz, T. Stueve, and S. Gallipeau. 2010 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9 

Citation: 

Hayes, T. B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. Adame, E. Chan, 

D. Bucholz, T. Stueve, and S. Gallipeau. 2010. Atrazine induces complete feminization 

and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of 

the National Academy of Science. www.pnas/cgi/10.1073/pnas.0909519107

Date of Review: March 3, 2010

Summary of Study Findings: 

Genotypic male (ZZ) African clawed frogs (Xenopus laevis) were crossed with 17-β

estradiol sex-reversed (phenotypic female) ZZ males plus ZW (genotypic/phenotypic) 

females and the resulting ZZ male larvae were exposed to atrazine at 2.5 μg/L from 

hatching through metamorphosis and for a period of up to 3 years post-metamorphosis. 

According to the study, atrazine-exposed males were both demasculinized and 

completely feminized as adults; 10% of the ZZ males (4 out of 40) developed into 

functional phenotypic females/genotypic males (confirmed by absence of DM-W gene 

marker in affected animals and their offspring) that mated with unexposed males and 

produced viable eggs. According to the study authors, atrazine-exposed ZZ males 

exhibited decreased plasma testosterone, decreased breeding gland size, 

“demasculinized/feminized” laryngeal development, suppressed mating behavior, 

reduced spermatogenesis, and decreased fertility relative to a solvent (ethanol) control 

males. 

Sex-reversed (ZZ) males (phenotypic females) were confirmed by absence of DNAbinding domain W (DM-W) marker gene. Researchers also relied on a morphometric 

analysis consisting of body weight and snout-to-vent length, presence/absence of nuptial 

pads and cloacal labia. 

Study Design: Study reports that there are 2 treatments (atrazine and ethanol control) 

with 9 tanks per treatment and 30 tadpoles per tank. Therefore, there are 270 animals per 

treatment (atrazine and ethanol control). Animals from three of the 9 tanks (representing 

90 animals) from each treatment were sacrificed at metamorphosis; however, according 

to Figure S1 (reproduced below) of the Supporting Information document, the sacrificed 

animals were used for the purpose of sex ratio analysis at metamorphosis. After 

metamorphosis, the remaining animals were then grouped in 3 tanks with 40 animals per 
91

tank (79x91x30 cm; 216 L) yielding 120 animals per treatment group (atrazine and 

ethanol control). Animals were reared to adulthood for 2 years in Holtfreter's solution 

under static renewal (72-hr) conditions in fiberglass tanks at 22o

C. The original source of 

the animals was from a feral stock collected in 1992 from Tecalote Creek in San Diego 

County, CA. Experimental animals were obtained by breeding the wild caught (feral = 

F0) males and females, all of which were injected with human chorionic gonadotropin 

(hCG). For analyses in this study, animals from the 3 tanks containing 40 animals per 

tank were divided into 38 tanks (46x25x20 cm; 23L) and raised singly (for fertilization 

experiments) or in groups of 4 (4 breeding competitions) while the remaining animals 

were used for undescribed studies. 

Figure 1. Figure S1 of the supplemental information from Hayes et al. 2010, depicting the "rearing 

program" for animals involved in the study. 

Atrazine levels in both treatment groups were monitored using ELISA; results of the 

ELISA assay are not reported. 

Aromatase activity: Reverse transciption-polymerase chain reaction (RT-PCR) was used 

to quantify cyp19 aromatase using RNA extracted from gonads of the two atrazinetreated females that were euthanized, four control males and four stock ZW females. 

Fertility analysis: In December 2007, 9 ethanol control and 9 atrazine-treated males (w/o 

hCG injections) were paired with stock ZW females (hCG injected). The next day, eggs 

were collected, aerated, and allowed to develop for 72 hrs. Fertility was determined by 

counting the number of developed embryos (NF stages 14 - 34). The study was repeated 

in December 2008 with 5 animals from each group (atrazine and ethanol control). The 

animals from both these analyses were the same ones used for histological and 

morphometric analyses. According to the study, atrazine-exposed males had significantly 

lower fertility rates (measured by the proportion of eggs fertilized) relative to control 

males. 

Histological analysis: Based on the largest testicular cross-sections for representative 

control and atrazine-exposed males from 2007, the control testicular tubules were 
92

typically filled with mature spermatozoa bundles, whereas the majority of tubules in 

atrazine-exposed males lacked mature sperm bundles and were nearly empty with 

secondary spermatocytes confined to the periphery of the tubule. Additionally, atrazine 

treatment altered the physical structure, but not the overall size of the larynx. 

Mate choice: 4 stock females (ZW) injected with hCG, 4 stock control males, and 4 

atrazine-treated males were placed together in 264 L of dechlorinated water; animals 

were left overnight and at 6 am the following day, and amplectant pairs were observed. 

Blood samples were then collected from pairs and the single males via cardiac puncture. 

Plasma testosterone was determined using RIA. According to the authors, these analyses 

were replicated 5 times; however, only 4 of the replicates were used since one of the 

replicates was omitted due to failure in the lighting system. As such, there were a total of 

16 females, 16 atrazine-treated males and 16 ethanol control males used in these 

analyses. There was no statistical difference in either snout-to-vent length or weight of 

the males used in the pairings. According to the study, control males had significantly 

higher plasma testosterone levels [in the presence of females] when compared to atrazineexposed males. Control males (11/16) out-competed atrazine-exposed males for 

amplexus with females; however, two atrazine-exposed males in a single trial achieved 

amplexus. 

 

Description of Use in Document: Invalid for survival, growth and metamorphosis due to 

lack of reporting results. Qual for sexual development, biochemical analyses and 

behavior. 

Rationale for Use: In general, the methods used in the study are scattered throughout 

the document and its supporting information. As such, it is difficult to understand the 

study design and how the design relates to the statistical analyses reported in the paper. 

For at least some analyses, judgment sampling rather than random sampling was used. 

At no place in the document do the authors state that random sampling was employed to 

avoid/reduce bias. The study relies on larval exposure conditions that have been 

previously reviewed by EPA and FIFRA SAP and deemed capable of introducing serious 

confounding effects on the study. Additionally, although the study “rearing program” is 

clearly depicted in the supplemental information referred to in the main publication, 

reported sample sizes associated with various measurement parameters are not consistent 

with the rearing program depicting the study treatments and replicates within treatment. 

In general, and under the conditions tested in this study, atrazine exposure resulted in 4 

animals out of 40 that fully differentiated as phenotypic female/genotypic male (ZZ) 

adults. While the 4 animals certainly represent 10% of 40, the total number of animals in 

the adult exposure segment of this study was 120. It is unclear whether the 4 feminized 

animals reflect 10% of a subsample or 3.3% of the total. While the authors state that the 

present study demonstrates atrazine exposed males were both demasculinzed (chemically 

castrated and completely feminized as adults, the data presented in the paper do not 

support such a global statement since the available data suggest that only 4 or 120 

animals were “completely feminized”. Given the uncertainties regarding the larval 

exposure conditions and their potential impact on gonadal development, the relevancy of 

statistically significant effects reported during the adult exposure phase of the study is 
93

also uncertain. Finally, there are a number of uncertainties/discrepancies in the figures 

used to depict study results. Additional information would be needed to resolve these 

discrepancies/uncertainties.

Limitations of Study: 

Animals in the initial breeding colony established in 1992 were wild-caught X. laevis

collected in Tecalote Creek in San Diego County, CA. The previous exposure history of 

the breeding colony was not documented in the study. The report does not state how 

many ZZ males were crossed with sex-reversed ZZ frogs to produce the larvae used in 

the study. This information would speak to the biological diversity that was represented 

in larvae subject to atrazine treatments. Additionally, it is uncertain how the limited 

parentage of an in-bred colony dating back to 1992 is reflective of the potential 

sensitivity of wild-caught animals from their native range in South Africa. 

Figure S1 (see Figure 1 above) of the supporting material depicts the “rearing program” 

for animals involved in the present study; however, it refers to the component elements of 

the rearing program as “treatments” and “replicates”. It is unclear whether this figure is 

also intended to represent the study design. In general, the study does not describe and/or 

account for all of the animals used in each treatment/replicate. As such, N values vary 

across measurement endpoints and it is unclear how measurement statistics relate back to 

the overall number of animals used in each treatment or to the study as a whole. 

Additionally, animals that were not part of the rearing program depicted in Figure S1

were also utilized in this study even though they were not reared and/or treated similar to 

either the solvent control or atrazine-exposed animals. 

According to Figure S2 of the Supporting Information document, ZZ male and ZW 

females were treated with estradiol and the resulting animals ZZ male/ZZ female and ZW 

female were crossed to produce 100% ZZ males. The resulting generation would have to 

be carefully proofed using the genetic marker to ensure that the study used exclusively 

100% ZZ males. According to the supplementary information, the ZZ females 

[presumably used in this study] were first generated in 1993. It is unclear from the study 

whether all of the parental stock animals used to produce larvae were subjected to 

estradiol 

The Nieuwkoop and Faber (NF) development stage of the larvae at initiation of exposure 

is not stated. The report simply states exposure initiated at hatching. An earlier study by 

Hayes et al. 2002 reported study initiation 4 days post-hatch (NF Stage 48); however, it is 

unclear whether the same protocol was followed for this study. 

The study reports that aquaria (29x18x13 cm; 6.8 L) were used for rearing larvae to 

metamorphosis; however, the aquaria construction material is not reported. Adult rearing 

tanks were constructed of fiberglass and it is uncertain how this construction material 

may have impacted water quality over the three-year study period. 
94

Exposure tanks are reported to contain 4 L of 10% Holtfreter’s solution with 0.003% 

ethanol. The initial study by Hayes et al. 2002 reports similar conditions except that 

0.004% ethanol was used. It is unclear why ethanol was required in the current study 

since no positive controls [with limited solubility] were used and the exposure 

concentration of atrazine (2.5 μg/L) used in the study is well below the chemical’s 

solubility limit of 33 mg/L (MRID 41379803). Although the study reports the use of a 

solvent control, it does not report the use of a negative control to determine whether the 

solvent had an effect. As such, the study does not account for the potential effect of 

ethanol. 

The study relies on a single exposure concentration (2.5 μg/L); therefore, it is not 

possible to determine whether there is a dose-response for any of the measurement 

endpoints to atrazine exposure. 

Loading rates of 30 larvae in 4 L (7.5 larvae/L) are the same as the values reported in the 

initial study by Hayes et al. 2002. Also similar is the static-renewal (72-hr renewal) 

exposure conditions. Similar to preceding reviews and concurrence by the FIFRA SAP, 

the loading rates coupled with static-renewal exposure conditions is in excess of that 

recommended by ASTM to maintain suitable environmental conditions for aquatic 

animals. It is unclear how the higher density of animals may impact the study. 

Purina® Mills Rabbit ChowTM was used to feed larvae during this study; the study does 

not report whether this feed was analyzed for any estrogenic activity and/or the presence 

of the test chemical. 

The methods section reports that weights and snout-to-vent length were “determined” as 

part of a morphometiric analysis. It’s unclear, though, when these measurements were 

actually recorded and/or tested for significance. As discussed below, the morphometric 

analysis was confined to 9 control and 9 atrazine-treated animals measured in December 

2007 and a second set of 9 from each group sampled in December 2008. 

Laryngeal weights (after fixation), laryngeal weight corrected for body weight, laryngeal 

length, laryngeal length corrected for body size, and the maximum cross-sectional area 

(transverse) of the dilater laryngis muscle are all reported as measured. Although no 

summary statistics are presented on weight, the authors report that there was no statistical 

difference (p>0.05) in the size of the larynx, but that there was a statistical difference in 

the structure. 

No effects on size at or time to metamorphosis are reported. According to the study, 3 of 

the 9 “reps” were sacrificed (90 animals) at metamorphosis, but no data are reported on 

these animals. Although these animals were reported used to determine sex ratio at 

metamorphosis, the study does not provide the results of this analysis. 

No data are provided from the ELISA assay used to monitor atrazine in both the ethanol 

control and the atrazine treatment. Therefore, claims that the animals were exposed to 

atrazine for up to 3 years at 2.5 ug/L are unsubstantiated in terms of the actual exposure 
95

level especially given the static renewal conditions of the study and the fact that tanks 

were aerated. 

For the larval stage of the study there are 270 animals in each treatment (9 tanks with 30 

animals/tank). At metamorphosis, animals in 3 of the 9 tanks (90 animals) were 

sacrificed presumably leaving 180 animals. The juvenile/adult studies were conducted 

using 3 tanks with 40 animals/tank (120 animals) in each of the two treatments. It is 

unclear what happened to 60 of the animals (180-120=60) and how the remaining 

animals were apportioned into the 3 tanks with 40 animals per tank. 

According to the study, the animals used for the fertility analysis, i.e., 9 control and 9 

atrazine-treated in December 2007 and 2008 were also used for the morphometric and 

histologic analyses. The study does not describe whether these animals were selected 

randomly or whether some form of judgment sampling was involved similar to the use of 

the completely feminized frogs for aromatase determinations. 

The supporting information states that recent metamorphs (NF Stage 66) were then reared 

for 2 years and then apportioned into 23-L tanks for experiments involving fertility and 

mate choice. It is unclear though how the experimental design and the 2-year postmetamorphosis exposure relates to the 3-yr exposure referred to in the study. 

Presumably, larvae would have completed metamorphosis within 3-months of study 

initiation just after the larvae hatched. Post-metamorphic juvenile animals would then 

have been raised to adulthood during the first year (Year 1) of the exposure study and 

exposures would have continued for 2 more years (Years 2 and 3); however, Year 3 of 

the study would not represent 3 years post-metamorphosis but rather 3 years post-hatch. 

Figure 1 of the paper depicts a series of photographs of ethanol control and atrazinetreated males and stock females. Additionally, some of the photographs depict atrazineexposed female (phenotypic female genotypic males). Without micrometer scales in each 

of the photographs, it is difficult to determine whether each of the structures depicted are 

at identical scales. Although the authors rely on brackets to indicate protruding labia, the 

interpretation of these pictures is subjective without evidence to indicate that the scales 

are identical. Although the authors attempt to emphasize differential development 

through the use of different-size brackets, the characterization does not appear to be 

based on any measurement. While the pictoral representations of control and atrazinetreated males may lend themselves to qualitative characterizations, such characterizations 

would be relatively subjective. More detailed image analyses and/or measured gonad 

weights would lead to a more objective and quantitative characterization of potential 

effects provided that the data were representative of the entire treatment group. 

Additionally, the comparison to animals (stock females) that were not subject to the same 

exposure conditions raises uncertainty. 

The relevancy of the two figures (Figures 1 G and H) depicting amplexus between 

ethanol control males and phenotypic female genotypic males is uncertain. 
96

Figure 3 of the paper depicts representative largest breeding glands (selected from the 

midpoint of the nuptial pads) from control (C) and atrazine-exposed (D) males. The area 

of the largest section of the largest gland was then determined for each sample and the 

bar graph adjacent to the micrograph depicts that breeding gland area (μm2

) as 

significantly less in atrazine-treated versus control animals. The micrometer scale in 

Figure 3D states that the scale is 0.1 mm or 100 μm. Applying this scale to the 

micrograph suggests that the cross-sectional area is minimally 220 x 180 μm or 39.6 

mm2

. As such, the breeding gland area in terms of μm2 depicted on the y-axis does not 

appear to be consistent with the micrometer scale. 

According to the study, the maximum cross-sectional area of breeding glands was 

determined and compared with the maximum cross-sectional area of mucous and serous 

glands. The results of these cross-comparison (breeding vs mucous vs serous) analyses 

are not apparent in the study. 

According to Figure 3, atrazine-exposed males had a laryngeal morphology intermediate 

between unexposed males and females. The transverse cross-sections depicted in Figures 

3F, 3G and 3H for ethanol control (ZZ) males, atrazine-exposed (ZZ) males and stock 

(ZW) females do not demonstrate a marked difference in the overall area for control and 

atrazine-treated males; however, the micrograph depicting the atrazine-treated male 

appears to be blurred. While the authors state morphologies were intermediate between 

ethanol control males and stock females, the intermediate nature of the atrazine-treated 

cross section is subtle at best. However, the authors employ a range of measurements 

and ratios to quantify the morphological differences that could not be quantified in terms 

of overall size. According to Figure 3, a sample size of 11 was used in this analysis; 

however, it is unclear how this sample size related to the overall study rearing program 

depicted in Figure S1 of the supplemental information regarding the study. 

The total number of animals later apportioned into 38 tanks, either as individuals or as 

groups of 4 is not stated. The study states that “remaining animals were used for studies 

not described here.” As such, it is unclear how many animals were exposed; therefore, 

the nature and extent of replication used in this study is uncertain. 

RT-PCR for cyp19 aromatase was conducted on only two of the atrazine-treated animals, 

i.e., two of the animals determined to be ZZ females. Additionally, four control (ZZ) 

males and four stock (ZW) females were included in the analysis for comparison. It is 

unclear why the aromatase assay was limited to only two animals in the atrazine-treated 

group if the intent is to make generalizations regarding the overall effects of atrazine on 

males to support the hypothesis that atrazine up-regulates aromatase. Additionally, the 

study incorporates animals (ZW females) that are not part of the study and as such do not 

reflect study conditions nor treatment with ethanol. In general, the number and scope of 

animals assessed is not consistent with the sampling design depicted in Figure S1 of the 

supplemental information associated with the publication. As such, it is unclear how the 

aromatase activity data generated from the assay can be related to the overall atrazinetreated versus solvent control animals in this study design. 
97

Given that X. laevis is sexually dimorphic and that four animals in a single atrazine 

treatment group were characterized as fully feminized, it is unclear why the researchers 

were unable to provide at least a qualitative estimate of the extent to which animals were 

fully feminized across all three tanks (replicates) treated with atrazine. As such, there is 

uncertainty whether the reported incidence of complete feminization (10%) is applicable 

across the entire study or whether only 4 animals overall were clearly feminized and the 

incidence is actually 3% (4/120). There is also uncertainty whether the 4 phenotypic 

females were from a single tank or were spread across the other replicate tanks. 

Previous studies by these researchers have reported mixed sex/intersex/hermaphroditism 

among atrazine-exposed animals; however, this study reports no such mixed gender 

animals. By implication, mixed sex animals either revert to their original gender (ZZ 

male) or they continue to differentiate as completely feminized (phenotypic females) 

genotypic males. The report does not contain sufficient information to determine 

whether mixed gender (phenotypic) existed; although the method section reports that sex 

ratio was measured in a subsample of juvenile frogs (and then not reported). It is unclear 

why this issue is not discussed and why researchers have not been able to consistently 

replicate the previous mixed gender results. 

Overall, there is uncertainty whether the continued reliance on husbandry/environmental 

conditions during larval exposure is impacting the study. 

In the mating choice study, it is unclear how animals were selected for sampling, i.e., 

whether the animals were selected based on judgment or random sampling. Additionally, 

it is unclear how 8 males to 4 females affect the process of mate selection and hormone 

secretion. While the sampling procedures used in this phase of the study were likely 

intended to minimize the extent of anesthesia required, suturing (marking the animals 

with thread) and cardiac puncture are relatively invasive procedures to perform without 

anesthesia and would likely contribute to stress levels among the study animals. As with 

other components of this study, the mate choice involved the use of animals (stock ZW 

females) that were not part of the study design. 

Additionally, on the mating pair studies, the report states that animals were allowed to 

mate overnight and that amplectant couples were identified the following day. It is 

difficult to draw any conclusions from what may have occurred overnight unless the 

animal activity was video recorded; the report does not state that continual observations 

(video recordings) were made. However, the available data [based on Figure 4A] 

indicate that in each of the mating studies there was at least 1 female that did not mate, 

but that in 3 of the four studies, control males were more frequently observed as 

amplectant. 

The caption of Figure 4 states that male size did not affect breeding success and that 

there was no difference in size between atrazine-exposed and control males; however, it 

goes on to say that in all of the trials, smaller individuals from controls out-competed 

larger atrazine-exposed individuals. 
98

The text states that control males had significantly higher plasma testosterone levels in 

the presence of females, when compared with atrazine-treated males; however, the study 

does not report means. Rather, Figure 4C of the paper provides a graphic representation 

of the testosterone data. According to the caption, solid horizontal bars show mean 

testosterone levels for successful males and open bars show the mean for unsuccessful 

males. It is unclear from the information provided whether the statistical analysis 

included both successful and unsuccessful males from each treatment or whether a more 

refined assessment was conducted. Figure 4C of the study suggests that the mean 

plasma testosterone levels between both control and atrazine-treated frogs that were able 

to achieve amplexus were not significantly different. 

There is uncertainty regarding the information depicted in Figure 5F of the paper. Based 

on the number of squares and circles depicted, there appear to be 5 controls (black 

circles) and 6 atrazine-treated (red circles) males in 2007. In 2008, there appear to be 3 

controls (black squares) and 4 atrazine-treated (red squares) males. However, according 

to Figure S1 of the Supplemental Information, there is an N=9 in 2007 and an N=5 in 

2008 provided that there was no overlap (points obscured by other points). Therefore, it 

is unclear whether the lower N value is a result of data truncation and/or censoring 

Additionally, the caption to Figure 5 indicates that no data are depicted for females that 

did not lay eggs. While female X. laevis induced with hCG may not always drop 

developed eggs, it is unclear why they would not drop any eggs. Also, in the caption to 

Figure 5 it states that the majority of [semeniferous] tubules lack mature sperm bundles 

and are nearly empty; however, Figure 5F that 6 out of 10 atrazine-treated animals had 

greater than 80% of the tubules with sperm. 

Although the authors cite a number of other studies in support of their current research, 

the authors’ interpretation of these other studies is not consistent with that of the EPA. 

Many of the studies cited have been reviewed by both EPA science staff and the FIFRA 

SAP and these reviews differ substantially from what is reported/summarized by Hayes et 

al. (2010). 

Primary Reviewer: Thomas Steeger, Ph.D., Senior Advisor, Environmental Risk 

Branch 4, Environmental Fate and Effects Division, OPP. 

 
99

3.20. Haridon J.L., M. Fernandez, V. Ferrier, and J. Bellan. 1993 

OPEN LITERATURE REVIEW SUMMARY 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 4324 

Haridon J.L., M. Fernandez, V. Ferrier, and J. Bellan. 1993. Evaluation of the 

genotoxicity of N-nitrosoatrazine, N-nitrosodiethanolamine and their precursors in vivo 

using the newt micronucleus test. Wat. Res. 27(5). 855-862. (ECOTOX 4324). 

Purpose of Review: Registration Review

Date of Assessment: 04/30/12 

Brief Summary of Study Findings: 

The objective of the study was to examine the genotoxic potential of atrazine, alone or in 

combination with sodium nitrite (NaNO2) as well as N-nitrosoatrazine (NAT) on 

amphibian larvae. Other chemicals were discussed in the paper but are not discussed in 

this review. 

Larvae, Pleurodeles waltl, (n=15) were placed into glass beakers containing 1.5L 

reconstituted water only or 0.3 ppm atrazine alone or with 0.45 or 140 ppm NaNO2 at 

20°C for 12 days. NAT (believed to be formed from atrazine) at 3.75, 7.5 and 15 ppm 

were also evaluated. Larvae were fed live Chironomus larvae and water and feed were 

replaced daily. Atrazine was dissolved in dimethyl sulfoxide (DMSO) and the 

concentration was never greater than 0.05mL/L. After 12 days, blood smears were 

obtained from each larvae and the number of micronucleated erythrocytes was 

determined. For atrazine, tests were conducted under standard conditions, under no lights 

and reduced pH and under conditions believed to induce NAT formation. For all tests, 

there was no effect on total number of micronucleated red blood cells (MN RBC) 

compared to the control. For NAT an increase in MN RBCs was significant at 7.5 and 15 

ppm but not 3.75 ppm. 

Description of Use in Document: Qualitative. 

Rationale for Use: While this study provides information on the genotoxic potential of 

atrazine, there are uncertainties regarding the test design and statistical analysis. 

Limitations of the Study: 
100

Limited water quality parameters were reported. While the study indicates that the study 

was conducted at the maximum concentration that did not induce detectable 

physiological disturbances for 12 days, survival was not explicitly stated. As a solvent 

was used (DMSO is not a recommended solvent) it is not clear if the study was conducted 

using a solvent control. As loading was not reported in terms of g/L, there is uncertainty 

if the loading exceeded the recommended value (0.5 g/L for static-renewal tests ASTM). 

The report indicates that 15 larvae were placed into each beaker and based on the Figures, 

a sample size of 15 was used for each treatment, suggesting that one replicate was used 

per treatment and the results were from pseudoreplicates. If only one replicate was used, 

potential replicate variability could not be observed. In addition, as there was observed 

effects for NAT, it is unknown what potential influence the use of pseudoreplication had 

on the results. 

Reviewer: Amy Blankinship, Chemist, Environment Risk Branch 3, Environmental Fate 

and Effect Division. 

 
101

3.21. Houck, A., and S.K. Sessions. 2006. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: E117318. Houck, A., and S.K. Sessions. 

2006. Could atrazine affect the immune system of the frog, Rana pipiens? Bios. 77 (4) 

107-112.

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

To evaluate the report for potential affects to amphibians from atrazine exposure. 

Date of Review: 11/29/11 

Summary of Study Findings: 

The objective of the study was to evaluate the effects of atrazine on the immune system 

of amphibians. It was hypothesized that exposure to increasing concentrations of atrazine 

would correlate with a decrease in the immune system (as measured by lymphocyte 

counts) in frogs. 

Methods 

In Trial 1, twelve [adult] Rana pipiens frogs were exposed to three atrazine 

(concentrations (0 (control), 1 and 10 ppb) for four weeks. In Trial 2, eleven frogs were 

exposed to the same concentrations for eight weeks. There were 4 replicates [reviewer 

assumed 1 frog/per replicate] for each concentration (except for 3 reps for control in trial 

2). Each replicate maintained in a separate glass jar with cheesecloth covering opening. 

Jars kept at room temperature and solution was changed every 3 days and jars rearranged 

at random. Following standard protocols, sheep red blood cells (SRBCs) were used as an 

antigen to induce immune response. After frogs were exposed for 4 or 8 weeks, SRBCs 

were injected intraperitoneally for five consecutive days. On the sixth day, the frogs 

were euthanized and spleens removed and lymphocytes were recovered. Cells checked 

for viability and examined using a hemocytometer. In trial 1, 9 of the 12 frogs were 

killed using 3 from each replicate and remaining three frogs used for hemolytic plaque 

assays (HPAs). In trial 2 all frogs used for hemolytic plaque assays. The HPAs were 

performed using tissue culture dishes and frog spleen cells. The number of plaques was 

counted and recorded. All results were analyzed using ANOVA (% values transformed 

using arc-sine-square root function). 

Results 

In trial 1 all animals survived; in trial 2 one frog died in the 1 ppb group (study discusses 

that death may be due to possible infection based on an acquired immunodeficiency). 
102

For trial 1, there was a significant difference (p<0.001) in the number of plaques 

(representing activated B-cells) (Figure 1). HPA plates from trial 2 did not produce 

scorable plaques. In trial 2, there were no significant differences in the mean % of 

lymphocytes or the mean total number of red blood cells and lymphocytes between 

groups (Figures 2 and 3). 

Figure 1. Average number (± standard error) of plaques seen after one month of treatment with atrazine (p < 0.001)

 Figure 2. The effect of atrazine on the mean percent of lymphocytes (± standard error) of Trial 2 as measured by a hemocytometer (p 

= 0.525).

 Figure 3. The effect of atrazine on the mean total number of red blood cells and lymphocytes (± standard error) of Trial 2 as measured 

by a hemocytometer (p= 0.272). 

Description of Use in Document (QUAL, QUAN, INV): 

Invalid 
103

Rationale for Use: 

Overall, there is a lack of reporting on certain parameters of the experimental design and 

culture as well as confounding issues in Trial 2 as frogs were not feeding in trial 2. In 

addition, there is uncertainty as to how the frogs in Trial 1 were analyzed and results 

presented. 

Limitations of Study: 

In general there is a lack of culturing and experimental parameter information. The 

source of the frogs and the food and water source are not reported, therefore, there is 

uncertainty in previous exposure of contaminants to the frogs, water and food. 

The preparation of the control and test concentrations was not reported; therefore it is not 

known if a solvent was used in the study. The study does not indicate if the 

concentrations of the test solutions were measured. Therefore, it is not known if the 

control represents a dilution water control or a solvent control. 

Overall, there is some confusion by the reviewer as to how many frogs were used for 

each endpoint for Trial 1. Based on the reported study design it appears that either a total 

of 12 or 11 frogs were exposed in Trial 1 or 2, respectively. In Trial 1, the reviewer is 

unsure of how the animals were used as the study indicates “In trial #1, nine of the twelve 

frogs were killed, using only three from each replicate, while the remaining three frogs 

were used as test frogs for hemolytic plaque assays (HPA) using a tissue culture dish 

instead of a slide chamber.” The reviewer is unsure what the nine out of twelve frogs 

were used for, but is assuming it was for HPA, perhaps using the slide chamber method 

instead of the culture dish as the remaining three frogs were used a test animals for the 

HPA using a tissue culture dish instead of a slide chamber. However, the methodology 

for the HPA using the slide chamber method is not described. As such, the reviewer 

assumes that the results presented in Table and Figure 1 represent a sample size of 3 frogs 

per concentration which represents the nine of the twelve frogs previously discussed; if it 

represents results using potentially different methods then there is uncertainty in the 

results. The reviewer is unsure if in Trial 1 cell counts of lymphocytes and red blood 

cells were conducted as they are not reported, however the study indicates that for Trial 2 

all frogs were used for HPA, using tissue culture dishes, and report results for 

lymphocytes and red blood cells. 

 In addition, the report indicates that most of the frogs did not feed during the two month 

treatment in trial 2 and therefore this observation confounds interpretation of any of the 

results for this trial. 

In addition, the study author indicates that while not significant the results in trial 2 for a 

similar pattern as the plaque results in trial 1 in that there is a decrease in immune cells 

with increasing atrazine concentration. Given that they reported that most of the frogs in 

trial 2 did not feed during treatment, interpretation of these results should be viewed with 

caution. 
104

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
105

3.22. Howe, G.E., R. Gillis, and R.C. Mowbray (1998). 

Open Literature Review Summary 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

PC Code: 080803

ECOTOX Record Number and Citation: 18805

Howe, G.E., R. Gillis, and R.C. Mowbray (1998). Effect of chemical synergy and larval 

stage on the toxicity of atrazine and alachlor to amphibian larvae. Environmental 

Toxicology and Chemistry, 17(3): 519-525. (ECOTOX 18805). 

Purpose of Review: Registration Review

Date of Review: March 1, 2012 

Summary of the Study: 

This study was conducted to explore the acute toxicity of field-grade formulations of 

atrazine, alachlor, and a 50:50 mixture of the chemicals to northern leopard frogs (Rana 

pipiens) (early and late stage), American toad (Bufo americanus) (early and late stage)76, 

rainbow trout (Onchorhynchus mykiss), and channel catfish (Ictalurus punctatus). The 

most sensitive 96-hr LC50 values for each species/chemical combination were as follows: 

- Alachlor: 

o Leopard frog (late) = 3.5 mg a.i./L 

o American toad (late) = 3.3 mg a.i./L 

o Rainbow trout = 9.1 mg a.i./L 

o Channel catfish = 16.7 mg a.i./L 

- Atrazine:

o Leopard frog (late) = 14.5 mg a.i./L 

o American toad (late) = 10.7 mg a.i./L 

o Rainbow trout = 20.5 mg a.i./L 

o Channel catfish = 23.8 mg a.i./L 

- Atrazine + Alachlor:

o Leopard frog (late) = 2.1 mg a.i./L 

 

76 Early stage = Gosner stage 29; late stage = Gosner stage 40. 
106

o American toad (late) = 1.5 mg a.i./L 

o Rainbow trout = 6.5 mg a.i./L 

o Channel catfish = 7.5 mg a.i./L 

Methods:

Leopard frog and American toad larvae were wild-caught from ponds known to be free 

from contaminants (i.e., no traces found). Larvae were acclimated to test water 

temperature for 1 week. During the acclimation period, the larvae were fed a 

combination of TetraminTM fish food and cultured Selenastrum sp. algae twice daily. 

Water flow rates were maintained at a minimum of one exchange of water/hr. The larvae 

were acclimated to the test water for a period of 24 hrs prior to the initiation of the test. 

At the onset of the test, early- and late-stage leopard frog larvae ranged in weight from 

0.7 – 0.9 g and from 1.4 – 1.9 g, respectively. Early- and late-stage American toad 

larvae ranged from 0.1 – 0.2 g and from 0.4 – 0.5 g in weight, respectively. 

Rainbow trout were obtained as eggs and channel catfish were obtained as fry from 

hatcheries; both were reared at the Upper Mississippi Science Center. At the initiation of 

the study, the rainbow trout ranged in weight from 0.8 – 1.0 g and channel catfish ranged 

from 0.9 - 1.1 g. Rainbow trout were held in 12o

C flowing well water for 96 hr prior to 

testing. Channel catfish were held in flowing well water that was gradually warmed from 

12 to 22o

C over a period of 96-hrs prior to testing. All fish were acclimated to the test 

water for a 24-hr period. 

Test chambers were glass jars filled with 15 liters of oxygen-saturated exposure water 

with an automated jar filler that was calibrated daily. During the study, mean water 

hardness was 44 mg/L as CaCO3, mean alkalinity was 33 mg/L as CaCO3, and mean pH 

was 7.9. The minimum oxygen concentrations exceeded 6 mg/L in all test chambers. 

Water temperature was maintained (± 1.0oC) at 12oC for rainbow trout and 22oC for all 

other species. 

Field-grade formulations of atrazine 4L (40.8% a.i.) and alachlor EC (43.0% a.i.) were 

weighed and mixed directly with deionized water to prepare the stock solutions. 

Exposure concentrations for each species were determined from range-finding tests 

(using a control and 5 concentrations). The definitive tests each consisted of 9 

concentrations and a control. Twenty individuals of each species (two replicates of 10 

each) were exposed for 96 hr to each selected concentration. The concentration of 

atrazine and alachlor in test waters was verified by immunoassay for the high and low 

exposure concentration of each test series. The concentrations remained within ±10% of 

the target concentrations throughout the study, except for two alachlor samples taken at 

96-hr for the channel catfish (they were 85% and 83% of the target concentrations). 

Nominal concentrations were used to calculate LC50s. 

Results:
107

The study resulted in the following 96-hr LC50s (see Table 1): 

TABLE 1. 96-hr LC50 Values for Alachlor, Atrazine and Atrazine + Alachlor for 

the Leopard Frog, American Toad, Rainbow Trout, and Channel Catfish. 

SPECIES (STAGE) LC50 VALUES (mg a.i./L) (95% C.I.) 

ATRAZINE ALACHLOR ATRAZINE + 

ALACHLOR

Leopard frog (early) 47.6 (41.4 – 54.8) 11.5 (10.1 – 13.2) 6.5 (5.7 – 7.7) 

Leopard frog (late) 14.5 (11.9 – 17.5) 3.5 (3.1 – 3.8) 2.1 (2.0 – 2.3) 

American toad (early) 26.5 (23.0 – 30.5) 3.9 (3.7 – 4.2) 1.8 (1.7 – 1.9) 

American toad (late) 10.7 (9.2 – 12.5) 3.3 (2.8 – 3.6) 1.5 (1.4 – 1.6) 

Rainbow trout 20.5 (18.3 – 22.9) 9.1 (9.0 – 9.2) 6.5 (5.7 – 7.7) 

Channel catfish 23.8 (22.3 – 25.5) 16.7 (15.1 – 18.4) 7.5 (5.3 – 8.4) 

The early stage amphibian larvae appeared to be less sensitive to the tested chemicals 

than the late stage larvae, and late stage amphibians appear more sensitive than fish to the 

chemicals. Additionally, the toxicity of the 50:50 mixture of atrazine + alachlor appeared 

significantly greater than additive at 96-hr of exposure for all test species and stages 

except the rainbow trout, indicating chemical synergy. 

The study authors postulate that late-stage amphibian larvae may be more sensitive than 

early-stage larvae due to the stresses associated with the onset of metamorphosis. 

Because of this, the authors state that acute toxicity data presented only for early-stage 

larvae should be interpreted with caution because they may in some cases underestimate 

hazards for later life stages. 

Description of Use in Document (QUAL, QUAN, INV): Qualitative 

Rationale for Use: This study provides mortality information for amphibians and fish 

from exposure to atrazine formulation 

Limitations of Study: 

- The acclimation period (1 wk) was shorter than the recommended acclimation period 

for fish (2 wk). 

- No raw data were provided. 

- The study was conducted using a formulation and not TGAI therefore there is some 

uncertainty if the toxicity observed was due to atrazine or an inert ingredient or some 

combination. 
108

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 Not applicable 

 
109

3.23. Kerby, J.L and A. Storfer. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Kerby, J.L and A. Storfer. 2009. Combined 

effects of atrazine and chlorpyrifos on susceptibility of the tiger salamander to 

Ambystoma tigrinum virus. Eco. Heal. . (ECOTOX Ref No. 121497) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/29/12 

Summary of Study Findings: 

The objective of the study was to examine the effects of multiple stressors of a viral 

pathogen (Ambystoma tigrinum (ATV)) and atrazine and chlorpyrifos on 4-week old 

tiger salamander larvae (Ambystoma tigrinum). 

Methods 

4-week old tiger salamander larvae were exposed in a fully factorial study: 3 

concentrations of technical grade atrazine [0, 20, 200 μg/L]; 4 concentrations of 

chlorpyrifos [0, 2, 20, 200 μg/L]; and either the ATV or not. Control treatments 

contained methanol solvent [reviewer calculated (1000 μL of 10% methanol solution in 

500mL = 0.2 mL/L)]. Larvae were reared in the laboratory obtained as eggs from 5 full 

sibship families at Arizona State University. There were 10 replicates (plastic cups; 

500mL solution per cup) per treatment with one larvae per replicate. Some replicates 

received the Ambystoma tigrinum virus (virus conc. at estimated LC50) via water bath for 

the first7 days of the test. Test solutions were renewed on day 7 and the study was 

conducted for an additional 2 weeks when death had subsided in the virus treatments for 

3 consecutive days. Animals were held at 20 ± 1oC. Survival was monitored daily and 

deceased organisms preserved immediately. Virus loads and infection status of larvae 

were analyzed using real-time PCR using tail tissue and measured in triplicate. All virustreated animals examined along with random sample of no-virus controls to verify lack of 

contamination. 

Results 

According to the authors, there was no significant main effect on mortality for atrazine, 

or interactive effects of atrazine by chlropyrifos on mortality. However, there was a 

significant interactive effects of atrazine by virus (p = 0.03) with atrazine slightly 
110

reducing mortality in no-virus 

treatments and slightly 

increasing mortality in virus 

treatments (Figure 2). 

Figure 2. Proportion of salamander larvae 

that survived viral and atrazine exposure. 

Pesticide levels span ecologically relevant 

concentrations and viral exposure level 

typically results in 50% survival. Logistic 

regression analyses are performed on total 

proportions, and therefore no standard error 

bars are present. Each bar represents all 

larvae in a particular treatment, and 

therefore combines larvae across all [atrazine] treatments

According to the study authors, the three-way interaction effect (virus, atrazine, 

chlorpyrifos) was not significant but the combined pesticide treatments resulted in the 

greatest mortality in virus-exposed animals (Figure 3). 

Atrazine had no effect on 

infection rate and no 

interactive effects between 

atrazine and chlorpyrifos on 

infection rate was detected. 

Results of viral load 

suggested that death 

corresponds strongly to 

infection in virus exposed 

treatments with higher viral 

loads in dead larvae than surviving larvae. Exposure to atrazine did not significantly 

affect viral load and analysis using infected only animals did not show effects in viral 

load with respect to atrazine [data not shown]. 

Figure 3. Proportion of salamander larvae that survived viral and pesticide exposure. Virus only data presented to show 

additive effects of increasing pesticide concentrations. Logistic regression analyses are performed on total proportions, 

and there no standard error bars are present.

Description of Use in Document (QUAL, QUAN, INV): QUAL 

Rationale for Use: 

While this study provides some information about survival of the tiger salamander from 

atrazine exposure as well as the possible interactive effects of atrazine and a virus on 
111

survival, there are uncertainties in the test design (no dilution water control, plastic test 

vessels) as well as the interpretation of the chemical’s role in mortality in the virus 

exposed larvae. 

Limitations of Study: 

A negative (dilution water only) control did not appear to be conducted in this study. 

Therefore, there is uncertainty was to what potential effect the addition of the solvent 

may have had on the results. In addition, based on reviewer calculations, the solvent 

concentration was 0.2mL/L which is 2 times the maximum amount recommended in 

OPPTS guidelines for chronic testing, but is within the 0.5mL/L limit for acute static 

testing. 

The larvae were maintained in plastic cups during the study; the type of plastic was not 

specified, but use of certain plastics are not recommended due to potential leaching. 

Dead larvae were preserved immediately upon discovery; however, as they were only 

observed daily, there may be some uncertainty as to the impact on viral load from 

potential differences in time of death before preservation. 

According to figure 2, the proportion of surviving organisms not exposed to atrazine or 

the virus (solvent control) appears to be about 75%; based on Figure 3, the control 

survival appears to be about 70% which is somewhat low. 

According to the study, the concentration of virus that the larvae received was the 

estimated LC50, and according to many of the results in the figures, the percent alive 

appears to be around 40-50% for many treatments. Therefore, there is uncertainty in the 

extent of the atrazine’s role in mortality for these treatment groups as it is assumed that 

the LC50 for the virus was calculated from a regression curve with multiple treatments 

and that there would be confidence intervals around that LC50. Therefore, while it does 

seem that there is a slight increase in mortality when exposed to concentrations of 

atrazine, one needs to consider the probable variability around the percent mortality from 

the viral exposure. 

The study authors state that in the three-way interaction the combined pesticide 

treatments resulted in greatest mortality. Based on the reviewers reading of Figure 3, for 

the 20 μg/L atrazine group with both the 2 and 20 μg/L chlorpyrifos group, the 

proportion alive was equal to or greater than the single concentration of chloryprifos or 

the single 20 μg/L atrazine group. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
112

3.24. Kiesecker J.M. 2002 

OPEN LITERATURE REVIEW

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 68326 

Kiesecker J.M. 2002. Synergism between trematode infection and pesticide exposure: 

A link to amphibian limb deformities. Proc Nat. Acad. Sci. 99 (15): 9900-9944. 

Purpose of Review: Registration Review

Date of Assessment: 4/20/12 

Brief Summary of Study Findings: 

This study encompassed both field and laboratory studies with three chemicals 

(malathion, esfenvalerate and atrazine). This review will focus on the laboratory study 

with atrazine. Wood frog embryos (Rana sylvatica) embryos (collected from three ponds 

in Centre County PA (two from each pond)) and hatched in outdoor pools and fed alfalfa 

pellets. Tadpoles (n=12) (presumed to be at Gosner stage 25 based on information in 

field studies; weight 0.112 ±0.012g)) were placed individually in polyethylene tubs in 

3.5L test solution at atrazine concentrations of 3 and 30 μg/L. Study indicated that 

DMSO or acetone were used to dissolve test material and therefore both a negative 

(dechlorinated tap water) and solvent control (% solvent in test solutions not provided) 

were used. Tadpoles kept at 16:8 light:dark cycle for 4 weeks and solutions changed 

every other day. After 4 weeks, tadpoles placed into 75 mL containers container either 

Ribeiroia sp or Telorchis sp cercariae (obtained from wild-caught snails) for 4 hours after 

which they were placed back into original containers under control conditions. After an 

additional week, blood was drawn from tadpoles for leukocytes enumeration, and then 

the tadpoles were euthanized and preserved for metacercarial cysts enumeration. 

The authors focused on the response of eosinophils because of the role they play in 

macroparasite infections. They state that exposure to pesticides resulted in decrease in 

eosinophils and all three pesticides (except for lowest exposure to malathion) had a 

similar effect on eosinophil numbers and proportion of cercariae that encysted (pesticide 

exposure p=0.006; pesticide x treatment p=0.320). In addition ‘pesticide exposure had a 

comparable effect on animals’ responses to the two different parasites’ (pesticide x 

trematode infection p =0.711) (Figure 1). 
113

 
114

Figure 1. Summary of effects of pesticides on eosinophils and % of cercariae 

successfully encysted (from Figure 2 in report) 

Description of Use in Document: Invalid 

Rationale for Use: A dilution water only and solvent only control, not exposed to 

atrazine or cercariae were not tested. Therefore, there is uncertainty in the response of 

the organisms in the absence of a stressor. In addition there are uncertainties in if 

cercariae were exposed to chemicals and uncertainties in statistical analyses. 

Limitations of the Study: 

A dilution water only treatment group was not tested. Analysis of the feed and water 

source for potential contaminants were not reported. Analyses of the test solutions for 

atrazine concentrations (including the controls) were not reported. The percent of solvent 

present in the solvent control or test solutions were not reported. Water quality 

parameters were not reported. Organisms were field collected, therefore their prior 

exposure is not known. Plastic containers were used as test vessels and plastic is not 

recommended. All test organisms were exposed to one sp. of the cercariae, therefore 

control groups (one negative and one solvent) that were not exposed to the cercariae to 

monitor organism performance were not used. Mortality in the study was not reported. 

There is uncertainty in how the statistical analyses were preformed and whether the 

atrazine treatment groups were compared to the negative control, solvent control or 

pooled control and exactly which treatment groups were significantly different (p<0.05). 

While based on Figure 2 in the study, there is a decrease in eosinophils in the atrazine 

groups and an increase in the proportion of cercariae encysting, there is uncertainty in 

exactly what treatment groups are statistically significant. 

 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
115

3.25. Kloas, W., I. Lutz, R. Urbatzka, T. Springer, H. Hrueger, J. Wolf, L. 

Holden, and A. Hosmer. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Kloas, W., I. Lutz, R. Urbatzka, T. Springer, 

H. Hrueger, J. Wolf, L. Holden, and A. Hosmer. 2009. Does atrazine affect larval 

development and sexual differentiation of South African Clawed Frogs? Trends in 

Compar. Endocrin. and Neuro.Ann. N.Y. Acad. Sci. 1163: 437-440. (ECOTOX Ref No. 

153612) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/27/12 

Summary of Study Findings: 

The objective of the study was to determine the potential transient impacts of atrazine on 

sexual differentiation processes in Xenopus laevis. 

Twenty-five sexually undifferentiated (stage 48) and differentiated (stage 58) Xenopus 

laevis tadpoles exposed to 25 μg/L atrazine and a control. Each treatment had two 

replicates as described previously in (Urbatzka 2007) and test solutions were replaced 

every 24 hours. After 4 days, 4 undifferentiated tadpoles were randomly sampled per 

tank (n=8). For the differentiated tadpoles, after 4 days exposure, they were randomly 

sampled from each tank until 4 males and 4 females were obtained (n=8/sex). Brain and 

gonads removed and several gene expressions for each were analyzed by quantitative 

RT-PCR. The gonadal anlage could not be separated from the kidneys in the 

undifferentiated tadpoles therefore mRNA analysis conducted on entire complex. Kidney 

and gonads were separated for sexually differentiated tadpoles. According to the study 

authors, in the testes, aromatase expression was negligible but had a high expression in 

the ovaries. The authors also state that for undifferentiated and differentiated tadpoles, 

gene expression was not different in the brain or kidney-gonadal complexes compared to 

control. 

Description of Use in Document (QUAL, QUAN, INV): 

Qualitative 
116

Rationale for Use: 

While this study provides some information on biochemical analyses several pieces of the 

test design and results are not provided. 

Limitations of Study: 

Preparation of test solutions, if used solvent and what constitutes the control are not 

specified. Water quality parameters, measurement of atrazine in test solutions, and 

survival not specified. As only two tanks were used per treatment but an n of 8 was 

reported in the result Figures, the analyses appeared to use pseudoreplication with 4 frogs 

from each tank for the undifferentiated tadpoles and four of each sex in the differentiated 

tadpoles. 

In the sexually differentiated tadpoles, there was negligible aromatase expression in the 

testes and high expression in the ovaries. As there was aromatase detected in all the 

kidney-gonadal complexes in the undifferentiated tadpoles, the study authors state the 

renal tissue contains aromatase. However, while the gonads were not wholly 

differentiated, the fact that the kidney-gonadal complexes did contain gonadal tissue and 

that aromatase was detected in the ovaries, and kidneys were not measured in the 

differentiated tadpoles to confirm aromatase expression, may it be possible that 

aromatase was present in the gonadal tissue as well as the kidney? 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
117

3.26. Kloas, W., I. Lutz, R. Urbatzka, T. Springer, H. Hrueger, J. Wolf, L. 

Holden, and A. Hosmer. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Kloas, W., I. Lutz, R. Urbatzka, T. Springer, 

H. Hrueger, J. Wolf, L. Holden, and A. Hosmer. 2009. Does atrazine influence larval 

development and sexual differentiation in Xenopus laevis? Toxicol. Sci. 107 (2): 376-

384. (ECOTOX Ref No. 112914) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/27/12 

Summary of Study Findings: 

This paper describes the Xenopus laevis larval development and gonadal differentiation 

study that was conducted under the DCI as a result of the 2003 SAP. This study was 

evaluated by the U.S. EPA and was brought forth in the 2007 SAP. Therefore, it is not 

necessary to summarize this paper. 

Description of Use in Document (QUAL, QUAN, INV): 

Not applicable 

Rationale for Use: 

Not applicable 

Limitations of Study: 

Not applicable 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

3.27. Koprivnikar, J., M. R. Forbes, and R. L. Baker. 2007
118

3.28. Koprivnikar, J., M. R. Forbes, and R. L. Baker. 2007 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Koprivnikar, J., M. R. Forbes, and R. L. 

Baker. 2007. Contaminant effects on host-parasite interactions: atrazine, frogs, and 

trematodes. Environ. Toxicol. Chem. 26 (10): 2166-2170. 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to examine whether atrazine reduces the defense of 

tadpoles to trematodes and if the reductions were behaviorally mediated. In addition, the 

study examined if atrazine increases susceptibility to parasitism. 

Rana sylvatica tadpoles were exposed to atrazine (40.8% formulation) concentrations of 

0, 3, or 30 μg/L (n=21) and Echinostoma trivolvis cercariae that were either also exposed 

to atrazine concentration or not (control cercariae). Tadpoles were obtained from egg 

masses that were collected from pond near Casselman ON, Canada; the authors stated site 

not tested for contaminants but pond receives no agricultural runoff. Tadpoles were 

individually exposed in 4L plastic tubs that contained 1.8L test solution that was renewed 

every 3 days and were maintained at 21oC for 31-days. Cercariae were obtained from 

snails that were field collected. For the experiment, tadpoles were placed into containers 

containing 50mL dechlorinated water and were exposed to 8 cercariae that were not 

exposed to atrazine. For experiment where cercariae exposed to atrazine, the cercariae 

were exposed to corresponding atrazine concentrations for 1 hour before being placed 

into container with tadpole. 6 tadpoles were used for each treatment group. For five of 

the six tadpoles exposed to atrazine but control cercariae, behavior observations were 

made by videotaping the organisms and recording activity. Tadpoles were exposed to the 

cercariae for 18 hours and then placed in groups of three into aquaria containing 

dechlorinated tap water. After 48 hours, tadpoles were dissected and examined for cysts. 

Results 

Survival in the 0, 3, and 30 μg/L groups was 61.9, 71.4, and 76.2% survivorship, 

respectively. Additionally, the study authors state that no effect was observed on the 

prevelance of infection between atrazine groups and control cercariae. There was a 
119

significant difference in cyst abundance and intensity between control and 30 μg/L group 

exposed to control cercariae. 

The authors indicate that the presence of control cercariae affected tadpole activity 

whereas atrazine concentration did not affect tadpole activity as well as no interaction 

was found between effects of cercariae and atrazine.

Description of Use in Document (QUAL, QUAN, INV):

INVALID 

Rationale for Use: 

Based on the study design, a control group that was not exposed to both atrazine and 

cercariae was tested whereas the control group exposed to cercariae had a mortality rate 

of 38%. 

Limitations of Study: A blank control was not used in the study in an effort to monitor 

tadpole performance of the population. In addition, replicate variability was not reported 

and effects were only observed when tadpoles, not cercariae were exposed to atrazine 

therefore, there is uncertainty in environmental relevance. Furthermore, limited water 

quality was reported 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
120

3.29. Koprivnikar, J. 2010 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Koprivnikar, J. 2010. Interaction of 

environmental stressors impact survival and development of parasitized larval 

amphibians. Ecol. Appl. 20 (8), p 2263-2272. (ECOTOX Ref No. 153870) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/23/12 

Summary of Study Findings: 

The study objective was to examine the interaction of trematode infection along with 

exposure to atrazine and predator chemical cues on the mass and development stage of 

northern leopard frogs. 

Methods 

Northern leopard frog tadpoles (Rana pipiens) were tested in a two-by-two-by-three 

complete factorial design with 32 replicates (one tadpole per replicate). The three factors 

were atrazine (0, 3, and 32 μg/L), Anax dragonfly larvae predation cue (present or 

absence), and Echinostoma trivolvis trematode parasite (cercariae) exposure (yes or no) 

for a total of 12 treatment group (excluding the negative and ethanol solvent control (100 

μg/L)). 

Tadpoles were obtained as eggs from Carolina Biological Supply Company (Burlington, 

NC) (six batches of eggs received and unknown if from same clutch) and raised in 

declorinated tap water and fed boiled spinach and rabbit chow. When tadpoles reached 

Gosner stage 25-26, they were individually transferred to 140-mL plastic cups; tadpoles 

without hind limbs (conducted using naked eye) and defined as < Gosner stage 27 were 

used for the study. Half of the cups contained cercariae obtained from naturally infected 

P. trivolvis collected from Santa Clara County CA. After cercariae exposure, tadpoles 

were individually transferred to a 2-L plastic container containing 1L test solution from 

either a negative or ethanol solvent (100μg/L) control, or 3 or 300 μg/L atrazine 

(99.2%). Test solutions were changed every seven days (report stated previous work 

indicated minor loss of atrazine over one-week period (Rohr et al, 2004)). Larval 

dragonflies (obtained from Ward’s Natural Science (Rochester, NY)) were placed into 

containers containing 0, 3 and 300 μg/L atrazine. Tadpoles were placed into containers 

to allow for predation by dragonflies. The dragonfly larvae were rotated through the 
121

three atrazine concentrations during 

successive predation events. Half of 

the experimental tadpoles received 

40mL of the appropriate 

concentration of strained predator 

water which was added the same day 

as the solutions were changed. Each 

of the experimental tadpoles were fed 

a pellet of rabbit chow each week 

and kept at 20°C with a 14:10 

light:dark cycle. Tadpoles were 

monitored daily until termination 

(when the first tadpole undergoing 

tail resporption was observed, ca.

G43). Tadpoles were euthanized, 

weighed and preserved; Gosner 

development stage determined along 

with dissections to enumerate the 

number of cysts in tadpoles exposed 

to cercariae. Four tadpoles from 

non-exposed cercariae were also 

dissected to look for kidney 

metacercariae. 

FIG. 1. Mortality of tadpoles of the northern leopard frog 

(Rana pipiens) under different treatments of (a) atrazine 

exposure and (b) exposure to a trematode parasite, 

Echinostoma trivolvis.

Results 

No significant differences between negative and solvent control, so data was pooled. 

Mortality in the 300 μg/L atrazine groups ranged from 95 – 100% and was not analyzed 

for interactions (Figure 1). Not all tadpoles exposed to cercariae became infected (n=26) 

and so data was analyzed by including and excluding the uninfected tadpoles. 

According to the study author, a significant difference among the three test 

concentrations was observed for survival; the model results from exposure to 3 μg/L 

atrazine (p=0.012) and exposure to atrazine and parasites (p=0.003) as well as exposure 

to parasites (p=0.013) and interaction between parasite and predator cues (p=0.007). 

Study author states that mortality was lower in parasite-exposed tadpoles but higher in 

tadpoles exposed to 3 μg/L atrazine; but that parasite exposed tadpoles experienced 

increased mortality when additionally exposed to 3 μg/L atrazine or predation cues while 

those not exposed to parasites did not (Figure 2). According to the data in Appendix A 

(Ecol. Arch. A020-085-A1), the percent mortality of tadpoles exposed simultaneously to 

3 μg/L atrazine, parasites and predator cues was 44%. For mass and development stage, 

the study author states that results including and excluding uninfected tadpoles negatively 

impacted developmental stage and that exposure to 3 μg/L atrazine decreased both mass 

and development stage. Exposure to 3 μg/L atrazine had a significant overall effect by 
122

decreasing both mass (p < 0.0001) and developmental stage (p< 0.0001) when parasite 

exposed but uninfected tadpoles were included or excluded. The author states that the 

significant interaction between parasite infection and exposure to 3 μg/L atrazine 

occurred because the mass of infected and uninfected tadpoles did not differ in the 0 μg/L 

group but the decrease in mass in the 3 μg/L group was stronger for infected tadpoles 

such that their mean mass was significantly less than that of uninfected tadpoles (Fig 3). 

Also the significant interaction between predator cue and 3 μg/L exposure occurred 

because predator cue-exposed tadpoles were significantly less developed than those not 

exposed to predator cues in the absence of atrazine, but was reversed in presence of 

atrazine, even though atrazine exposure negatively impacted development stage for both. 

FIG. 2. Tadpole mortality due to (a) parasite and predator 

cue (larval Anax dragonflies) exposure, and (b) parasite and 3μg/L atrazine exposure. Notations of – and+ indicate 

absence and presence, respectively, of exposure to factor.

The study author states that exposure to parasitism had little overall effect on larval 

amphibians in this study, however it is clear that this stressor in combination with others 

can have interactive effects and such be a consideration when considering impacts of 

pathogens and other stressors. 
123

FIG. 3. (a) Mean mass (untransformed) of parasite-infected(+) and uninfected (-) tadpoles in the presence of 

3 μg/L atrazine (dot-dashed line) or 0 μg/L atrazine (solid line). (b) Mean Gosner developmental stage of 

tadpoles exposed (+) or unexposed (-) to a predator cue in the presence of 3 μg/L atrazine (dot-dashed line) 

or 0 μg/L atrazine (solid line). Differentiation of the hind-limb buds begins at Gosner stage 31, and forelimbs 

emerge at Gosner stage 42. Error bars represent ±SE. Parasite-exposed but uninfected tadpoles were not 

included for either measure.

Description of Use in Document (QUAL, QUAN, INV): 

INVALID 

Rationale for Use: 

The variability in mortality across the different treatment groups raises uncertainty in the 

ability to attribute other effects (e.g., metamorphosis, growth) to the addition of atrazine.

Also, based on uncertainty in reported results based on Figures in report 

Limitations of Study: 

The tadpoles were maintained in plastic cups during the experiment therefore, chemicals 

from the cups may have leached into the test solutions. Also, the study did not indicate if 

the feed was free of potential contaminants. Water quality parameters were not reported 
124

As this study was conducted with two atrazine concentrations and one of them resulted in 

ca. 100% mortality, the reviewer could not determine a dose-response for the sublethal 

endpoints. 

Approximately 3% of the tadpoles that were not exposed to the parasite (dilution water 

only) died during the parasite exposure phase. When combining parasite exposed and not 

exposed tadpoles, 7.5% died overall during this phase, but it is hard to discern if exposure 

to the parasite caused the mortality as 6% of tadpoles exposed to only the parasite during 

the study died. However, as mortality at the preliminary exposure phase was observed 

there is some uncertainty in the fitness of the tadpoles. 

Determination of the starting Gosner stage was done with the naked eye (no obvious hind 

limb bud), therefore there could be some uncertainty in the actual stage for each of the 

tadpoles. 

Mass was an endpoint evaluated in this study and the study was terminated when the first 

tail resorption occurred. Based in the reported stage results, frogs were at different stages 

at test termination. Given that mass is correlated with metamorphosis stage for frogs, the 

reviewer is uncertain in how the study author accounted for this when comparing 

treatment groups. 

The reported results in the text and figures in this paper are difficult to interpret. From 

the figures and text, it is not certain whether atrazine alone significantly impacted 

survival. An example of difficulty in interpretation is, in Figure 2a, it is difficult to 

interpret what the figure is representing based on the figure title and the resulting bars. 

As it reads now, it appears that in the absence of predator and parasite, the % mortality is 

almost 40%. The reviewer is not sure if the codes for the parasite axis are switched 

because, if they were switched, then this appears to better correlate with the % mortality 

values reported in Appendix A as, for example, presence of both predator and parasite 

result in ca. 25% mortality which is the exact value reported in the Appendix. However, 

there is uncertainty in what the back corner bar represents because it does not appear to 

correlate with values in Appendix A. Therefore, the reviewer is unsure if this figure is a 

compilation of treatment groups or meant to represent individual treatment groups. This 

also appears to be true for Figure 2b, in which the absence of atrazine and parasite result 

is ca. 33% mortality. Also, the way the data is presented in the text can also be difficult 

to interpret and a clear indication of significant effects for which treatments in table form 

for example would have been helpful. 

Based on the values reported in Appendix A, there appears to be some variability in the 

mortality response for the tadpoles. According to Appendix A, mortality in the pooled 

controls was 16%. While the mortality of tadpoles exposed only to 3 μg/L atrazine was 

31%, tadpoles exposed to both atrazine and predator cues only exhibited 3% mortality. 

Therefore, the mortality of tadpoles exposed to 3 μg/L atrazine does not seem to be 

consistent, as it appears that the addition of the predator cues reduced mortality which is 

not the expected response. In addition, the mortality of tadpoles exposed only to predator 

cues was also 31%, while exposure to parasite and predator cues resulted in 9% mortality. 

While tadpoles exposed to both atrazine and parasites and to all three stressors had 25% 

and 44% mortality, respectively, given that tadpoles exposed only to predator cue had 
125

31% mortality and the variability in mortality between atrazine exposure and predator 

cues, it is difficult to conclusively determine that atrazine negatively impacted tadpole 

mass and developmental stage. Also, if mortality was significantly impacted at 3 μg/L as 

it appears to be based on the discussion, it may have influenced evaluation of the other 

endpoints given that the frogs may have expended energy on detoxification rather than on 

growth and development. However, the study author states the smaller tadpoles were 

likely not selected against due to the mortality as a significant decrease in mass was still 

observed. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
126

3.30. LaFiandra, E.M, K.J. Babbitt, and S.A. Sower. 2008 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: LaFiandra, E.M, K.J. Babbitt, and S.A. 

Sower. 2008. Effects of atrazine on anuran development are altered by the presence of a 

nonlethal predator. J. Toxicol. Environ. Health, Part A. 71: 505-511. (ECOTOX 

103336) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to examine the effects of a nonlethal predator and 

exposure to atrazine and their interaction on amphibian growth and development. 

Hyla versicolor tadpoles (Gosner stage 25) were exposed to either 0, 20 or 200 μg/L 

atrazine (Aatrex Nino-O, 88.2% atrazine and 1.8% related compounds) and exposure or 

not to a nonlethal predator, dragonfly larva (Anax sp) which was placed in a mesh cage in 

aquaria with tadpoles; tadpoles not exposed to nonlethal predator contained an empty 

cage. Tadpoles were obtained from a pair of adults collected while in amplexus (location 

not specified). Three tadpoles were maintained in glass tanks containing 7L test solution 

at 26°C with a 50% renewal weekly. Tadpoles remained in tanks until front limb 

emergered at which date of initiation of metamorphosis was recorded and then animals 

moved to individual containers. Following complete metamorphosis, frogs were 

weighed, snout-urostyle length measured and external deformities assessed after which 

they were maintained for approximately 1 month to allow for necessary size for 

dissection. Both gonads removed and preserved for histology. 

Measured concentrations were 16 and 195 ppb for the 20 and 200 ppb group, 

respectively. Other degradation products were measured, but it is not known if they were 

from the formulation or present in the dilution water. The proportion of tadpoles 

reaching metamorphosis was 75% in the 200 ppb group and was 95.8% in the control. 

The presence of the nonlethal predator significantly reduced the proportion of tadpoles 

that initiated metamorphosis at 200 ppb. Survival to dissection not affected by atrazine 

but was significantly reduced from 69.4% in no-predator group to 55.6% with predator. 

Larval period did not differ among predator treatments and no effects growth were 

observed. See figures below reprinted from study. According to the authors “Ovaries from 

females exposed to 200 μg/L atrazine were composed largely of previtellogenic oocytes, 
127

whereas females exposed to 20 and 0 μg/L atrazine contained ovaries consisting of nearly 

equal proportions of oogonia and previtellogenic oocytes. The impact of predatory stress 

on testicular development was only apparent in the atrazine exposures, and was most 

pronounced in the 200 μg/L exposure. However, the interaction effect was not 

statistically significant.” 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: the study provides information about interactions of atrazine and 

predator on amphibian development however, there are limitation 

Limitations of Study: 

Limited water quality was reported and there is uncertainty if adults used to obtain eggs 

for culturing were from the field or laboratory. 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. Degradates were 

measured in atrazine solutions and since control solutions were not reported there is 
128

uncertainty in whether the detected degradates were present in the formulation or dilution 

water.

Primary Reviewer:Amy Blankinship, Chemist, Environmental Risk Branch 3, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 
129

3.31. Langerveld, A.J., R. Celestine, R. Zaya, D. Mihalko, and C.F. Ide. 

2009. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Langerveld, A.J., R. Celestine, R. Zaya, D. 

Mihalko, and C.F. Ide. 2009. Chronic exposure to high levels of atrazine alters 

expression of genes that regulate immune and growth –related function in developing 

Xenopus laevis tadpoles. Environ. Res. 109: 379-389. (ECOTOX Ref No. 121497) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/29/12 

Summary of Study Findings: 

The objective of the study is to evaluate effects of atrazine on gene expression, growth 

and mortality in X. laevis. 

Methods 

Xenopus laevis tadpoles (stage 43 (5-days post-fertilization), tadpoles obtained from 

adults reared at laboratory) were exposed to 400 μg/L atrazine or a water control in a 

semi-static system. New solutions were prepared every 2-3 days; water temperature 

measured before every renewal (range 20-22°C). 15 replicates (glass dishes containing 

1L solution) were used for the atrazine group and 20 for the control with no more than 4 

tadpoles per replicate; the starting number of tadpoles was 78 for control and 58 for 

atrazine. According to the study authors, the original test design was to collect tadpoles 

at stage 66 to compare to other studies, however, in the atrazine group, tadpoles entering 

stage 63 or higher was dying at high rate. Therefore, 29 and 25 surviving tadpoles at 

stage 62 in addition to dead stage 62 tadpoles (1=control; 8=atrazine) were evaluated. 

Tadpoles found dead at stage 62 were evaluated for gender, gross morphological 

abnormalities. Endpoints measured for surviving stage 62 tadpoles include: weight, 

snout-to-tail length, and gross morphological (gender, fat bodies) or molecular analysis 

(gene expression); in tadpoles used for gene expression, fat bodies not measured and 

gender determined without Bouin’s fixative. Total RNA was isolated from 6 male 

control and atrazine tadpoles, 6 female control and 5 atrazine tadpoles. Samples were 

prepared for microarray according to Affymetrix GeneChip® protocols. Five genes were 

chosen from the final Affymetrix dataset for validation using Taqman qRT-PCR: 

pancreatic trypsin, chitinasse, chitobiase, neurohormone-like peptide and magainin II 

copy E. 
130

Results 

Survival in the 400 μg/L treatment group was staticially significant from the control; 

while no gross tissue changes 5 of 8 dead atrazine frogs had “small/underdeveloped” fat 

bodies. Male and female atrazine-exposed tadpoles were significantly shorter and 

weighed less than controls; animals analyzed by gender. Male and female tadpoles 

exposed to atrazine had a significant reduction in fat body area compared to control. 

According to the authors, chronic atrazine exposure altered expression of 44 genes in 

male tadpoles and 77 genes in female tadpoles and that the genes that were altered were 

involved in proteolysis and/or digestion, carbohydrate metabolism, growth, immune 

system functions, oxygen transport, heme binding and blood coagulation. However, the 

majority of genes were altered exclusively in either male or female tadpoles. For 

females, qRT-PCR showed a significant decrease for levitide, magainin, chitobiase and 

significant increase in trypsin; for males the analysis showed significant increase in 

chitinase. 

Description of Use in Document (QUAL, QUAN, INV): 

QUAL for survival; INVALID for other endpoints due to significant mortality observed 

at only atrazine concentration tested. 

Rationale for Use: 

This study provides information on survival for Xenopus laevis tadpoles at a single 

concentration. However, as some environmental condition information is missing there 

is some uncertainty in whether atrazine alone was the source of the mortality. 

Limitations of Study: 

The study authors refer to this test as chronic testing and the tadpoles move through many 

developmental stages, however the exact duration of the study is not reported. 

Statement if food and water were analyzed for contaminants or other possible 

interferences not provided. 

Analysis of test solutions for atrazine were not reported. 

While the loading in the test vessels was not reported, the study did indicate that no more 

than 4 tadpoles were placed into each vessel which contained 1 liter solution. Based on 

the graph in Figure B, the mean body weight of stage 62 tadpoles appears to range from 

ca. 0.35 – 0.48 g [reviewer visually determined this range from figure]; if there were 4 

tadpoles/vessel then the mass/vessel would exceed the recommended 1 g or 1 tadpole/L. 

Environmental conditions other than temperature were not reported. Therefore, 

information on conditions such as dissolved oxygen, pH and ammonia would be helpful 

in evaluating potential causes for the observed mortality. 

The total number of initially exposed in each treatment group is unclear and the total 

number of dead tadpoles from the total number that were exposed is unclear. The study 
131

indicates 15 dishes for the atrazine group and 20 dishes for the control were set-up and 

that each dish contained no more than 4 tadpoles, for which the reviewer assumed for a 

potential maximum of 80 and 60 exposed tadpoles. It also states that full datasets were 

maintained for 78 and 58 tadpoles for the control and atrazine group, respectively 

(reviewer presumed that not all dishes contained 4 tadpoles and that this was the initial 

number of exposed animals). The study states that as tadpoles were entering stage 63 

and higher, tadpoles were dying at a high rate in the 400 μg/L atrazine group; the study 

goes on to describe the number of tadpoles excluded and those included in the analysis. 

If one presumed that the tadpoles that were not at stage 62 and excluded survived, then 

that would result in one overall death in the control and 8 overall deaths in the atrazine 

group; however these deaths were all reported to be for stage 62 tadpoles and therefore 

do not appear to take into account any deaths from tadpoles entering stage 63 or higher as 

previously noted. Therefore, it appears that there must be some mortality in the excluded 

tadpoles to account for the statement that tadpoles entering 63 or higher were dying, but 

the number of mortalities are not reported. The reviewer assumes that the overall 

mortality in the control is within acceptable limits. 

As survival was affected in the 400 μg/L atrazine group, it is not wholly unexpected that 

other sublethal effects such as growth and gene expression would also be affected. Due 

to the lack of testing other non-lethal test concentrations, evaluation of these sublethal 

effects in the absence of mortality could not be made in this study. Testing only one 

atrazine concentration precludes the ability to discern a dose-response relationship. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
132

3.32. Langlois, V.S., A.C. Carew, B.D. Pauli, M.G. Wade, G.M. Cooke, V.L. 

Trudeau. 2010 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Langlois, V.S., A.C. Carew, B.D. Pauli, 

M.G. Wade, G.M. Cooke, V.L. Trudeau. 2010. Low levels of the herbicide atrazine alter 

sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor 

mesocosms. Eviron. Heal. Perspec. 118 (4): 552-557. (ECOTOX Ref No. 120046) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/27/12 

Summary of Study Findings: 

The objective of the study was to determine whether low concentrations of atrazine 

affects gonadal development and metamorphosis in the Northern leopard frog. The 

hypothesis was that exposure to atrazine alters estrogen receptor (α) (eralpha) mRNA 

level and 5β-reductase (srd5beta) activity in the target tissues of exposed tadpoles. 

Methods 

Northern leopard frogs (Rana pipiens) were collected in the Raisin River region, 

Cornwall, Ontario, Canada (reference site). The field survey (summer 2006) used youngof-the-year metamorphs (n=30) from the reference site; animals were euthanized, 

phenotypically sexed (visual observation) and the kidney-gonad complex was removed 

and fixed for histology. Atrazine (n=2/site per date) was measured from five different 

locations in the reference site every week for 5 weeks (May 25-June 19). 

In the mesocosm studies, tadpoles (Gosner stage 25 (G25); n=30, 6 tadpoles from each of 

five different egg masses collected from reference site and reared in laboratory at density 

of 1 g tadpole/L) were added to each microcosm at density of 0.1 g tadpole/L. 

Mesocosms (aged 378-L high-density polyethylene tank) contained 300L groundwater, 

rabbit pellets, dried leaves and Daphnia magna from local creek. Each cosm constantly 

aerated and covered with a shade cloth (nylon lid). When tadpoles at G27, exposed to 0, 

0.1, 1.8 μg/L atrazine (reported as formulation AAtrex Liquid 480 with a purity of 

97.1%), 1.5 μg/L EE2 and 0.0003% ethanol (EtOH) (for the EE2) (all treatments n=5). 

Atrazine was applied twice (May 12th and 19th). Dissolved oxygen, pH, and temperature 

recorded weekly; when tested, ammonia levels below detection (250 μg/L). Atrazine 

concentrations in mesocosms measured when spiked with atrazine and then roughly 
133

every 15 days [reviewer based on figure provided in supplemental information]. Rain 

samples collected onsite analyzed for atrazine were below detection (0.003 μg/L). When 

animals reached G42 (metamorphic climax), they were removed and recorded age at 

metamorphosis (AAM; days), wet weight, and snout-to-vent length (SVL). Sex was 

visually determined, and brain, liver, and tail removed and samples frozen; kidney-gonad 

complex fixed. Subset of male samples histologically analyzed for presence of testicular 

oocytes (serially sectioned longitudinally; blind analysis). At end of experiment, 

premetamorphic G34 tadpoles were analyzed for gene expression analysis. Real time 

RT-PCR was used to detect transcripts for cyp19, eralpha, TH receptor isoforms (tralpha

and trbeta), deiodinases 2 and 3 (dio2 and dio3, respectively, and ribosomal protein L8 

(rpl8), and neuropeptide corticotrophin-releasing hormone (crh); samples were conducted 

in duplicate. Enzymatic activity of cyp19 and srd5beta was conducted using radiometric 

methods. Endpoints for atrazine groups and EtOH control were compared to control and 

the EE2 group was compared to the EtOH control. 

Results 

The concentration of atrazine in the field samples (streams in the Raisin River Basin) 

ranged from 0.01 to 1.6 μg/L with detects in every stream sampled. For the mesocosm 

studies, the concentration of atrazine was <0.003 – 0.028, 0.09-0.21, and 1.6-3.7 μg/L for 

the control, 0.1 and 1.8 μg/L treatment groups, respectively. Based on Supplemental 

figure 2, it appears that the mean atrazine concentration was ca. 0.15 and 3 μg/L 

[reviewer observation]. Study authors state that possibly contaminated ground water 

used to keep mesocosm water levels up accounted for detects in control. No statistical 

difference in pH, temperature or dissolved oxygen. According to the study authors, 

survival was significantly lower for the 1.8 μg/L atrazine group and the EE2 group when 

compared to appropriate control; also, significantly less tadpoles reached metamorphosis 

in both atrazine groups and the EE2 group when compared to the control (Table 1). 

There was no difference in AAM, weight or SVL between controls and treatment groups. 

The sex ratio for the 1.8 μg/L group was significantly different from control with more 

females than males (1:1.14 vs. 1:0.6) with no differences for the other treatment groups 

(Table 1). The males in the EE2 group had 22% testicular oocytes (4 out of 18), with 

none observed in any other group. 

Table 1. Survival,Metamorphic Success, Sex Ratio for R. pipiens exposed to Atrazine or 

EE2 (reproduced from Table 1 and 2 in study report). 

Treatment Survival- # 

survived/total 

exposed (%) 

Metamorphic 

success (%) 

Sex Ratio (M:F) 

Water control 118 / 150 (79) 90 (76) 1:0.6 

0.1 μg/L atrazine 113 / 150 (75) 50 (45)* 1:0.8 

1.8 μg/L atrazine 99 / 150 (66)* 47 (50)* 1:1.14* 

EtOH control 114 / 150 (76) 99 (85) 1:0.6 
134

1.5 μg/L EE2 97 / 150 (65)** 52 (55)** 1:0.9 

Reference site -- -- 1:0.5 

* = p ≤0.05 compared to water control; ** = p ≤ 0.05 compared to EtOH control 

Atrazine did not significantly affect cyp19mRNA level or activity in the brain of the G42 

animals but EE2 did; also no changes in cyp19mRNA levels in brains of G34 tadpoles. 

In the brains of the G34 tadpoles, expression of eralpha in the 1.8 μg/L group was 2.5 

times greater than control, but no difference in the brains of G42 tadpoles or livers from 

G34 and 42 tadpoles or in EE2 group for any tissue or stage. There was a decrease of 

dio3mRNA in tail tissue at 1.8 μg/L atrazine for G34 tadpoles. No other changes were 

detected for the other gene endpoints, except for srd5beta. In the control animals, the 

females expressed 43% more srd5beta than the males, but no difference was observed 

between the sexes for the atrazine groups. 

Description of Use in Document (QUAL, QUAN, INV): 

QUAL 

Rationale for Use: 

The study provides some information on the survival and metamorphic success of the 

northern leopard frog, and 5 mesocosms per treatment allow for greater statistical power. 

However, there are some uncertainties regarding source of organisms, contaminated 

controls and overall conclusions. 

Limitations of Study: 

 Frogs used in the study were collected from a reference site that was surrounded by 

streams in which atrazine was detected (range 0.01-1.6 μg/L); therefore there is some 

uncertainty regarding previous exposure to atrazine and other potential contaminants. In 

addition, several sources of food and water were provided to animals and the report made 

no statements about possible contaminants in those sources. 

The study indicated that the formulation AAtrex Liquid 480 was used and reported the 

percent active as 97.1% atrazine and 2.9% related triazines but do not report the 

percentage of atrazine in the formulation [reference on Sygenta Canada website indicates 

it is 43.7%, 

http://www.syngentafarm.ca/prodrender/index.aspx?nav=labels&ProdID=778 ] 

The water control treatment group had measurable amounts of atrazine (up to 0.028 μg/L 

which is ca. 1/3 the lowest treatment group) during some portion of the test which that 

study authors hypothesized that may have come from contaminated groundwater for at 

least some portion of the study duration. 

It appears that according to the study, the tadpoles were placed into the mesocosms at 

stage 25 and exposure to the chemicals started when tadpoles were at stage 27. The study 

did not report how observations of the frogs was conducted to determine when at stage 27 
135

as well as how observed during the exposure and if survival/mortality was only 

determined at test termination. 

Survival and metamorphic success were reported based on total number of organisms 

exposed and not as a replicate mean with error bars reported; therefore, variability 

between replicates is not known. While no significant differences between groups during 

the study, there was some noticeable variation in temperature on at least one occasion 

(sampling point 7 on Supplemental figure 3) that seems to indicate a potential 4°C 

difference in the mean temperature between the water control and 1.8 μg/L atrazine 

group with a higher temperature in the atrazine group. Also, based on Supplemental 

figure 3, it appears that the dissolved oxygen dropped below 60% for at least two 

sampling time points (points 4 and 8). 

Except for the 1.8 μg/L atrazine group, there were more surviving males than females 

with or almost as twice as many males as females in the controls and the reference site. 

As there was mortality in control and treatment groups (21% in water control and 25 and 

34% in the 0.1 and 1.8μg/L atrazine groups, respectively) there is uncertainty in whether 

there was a differential sensitivity in survival between the sexes. In Table 1, survival was 

based on animals that reached or passed G42 (stated to be metamorphic climax), however 

in Table 2 only G42 individuals were used for gonadal gross morphology which resulted 

in fewer animals being used for sex determination based on the numbers presented in 

Table 1 and 2. The reason for only presenting sex ratio data on frogs at G42 was not 

stated in the study. The study states that animals were removed and processed when they 

reach G42, therefore it seems that every frog that that underwent complete 

metamorphosis should have been able to be sexed. The reviewer assumes it is that not all 

frogs were able to be removed at exactly G42 as some frogs beyond G42 are presented in 

Table 1. As sex ratio is presented as a subset of animals that completed metamorphosis 

there is uncertainty in how this affected the results. 

The study authors state that this study provides evidence that atrazine may feminize R. 

pipiens when exposed to low levels of atrazine and that they suggest that associated 

changes in brain eralpha mRNA level and liver srd5beta activity might be involved in 

producing the response. Now while the concentration of EE2 may not have been high 

enough, EE2, which was used as a positive control for feminization did not show any 

difference for eralpha mRNA based on the results section and Figure 2. However, in the 

Discussion section, the study authors state that in their mesocom study, EE2 doubled the 

brain eralpha mRNA level, suggesting that estrogenic compounds have the ability to 

increase eralpha mRNA levels. Results for liver srd5beta for EE2 not discussed. In 

addition, a dimorphism effect for srd5beta was observed between sexes in the control but 

not the atrazine groups. While the study authors state that there is data lacking in the 

importance of this dimorphism, the sex ratio for the 0.1 μg/L atrazine group was not 

significantly different than the control; therefore there is uncertainty in the role srd5beta 

may play in this study. The report did not indicate if there were any differences within 

sex across the treatment groups for srd5beta, but based on the reviewer’s interpretation of 

Figure 3, there does not appear to be. 

The authors also state that they present information that atrazine alters the thyroid axix by 

affecting metamorphosis success and also TH-related gene expression by a reduction in 

dio3 mRNA in G34 tadpole tails. While this reduction was significant in the 1.8 μg/L 
136

atrazine group it was not in the 0.1 μg/L group which also had a significant decrease in 

metamorphic success. 

Overall, there is some evidence that atrazine did affect survival of R. pipiens at 1.8 μg/L 

and may affect metamorphic completion at 0.1 (nominal) and 1.8 (nominal) μg/L. 

However, there is uncertainty in the results underlying metamorphism and the difference 

in sex ratio in this study. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
137

3.33. Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and 

Hamilton, S. J. (1998) 

OPEN LITERATURE REVIEW 

Chemical Name: Atrazine 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 60632 

Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and Hamilton, S. J. (1998). 

Effects of the Herbicide Atrazine on Ambystoma tigrinum Metamorphosis: Duration, 

Larval Growth, and Hormonal Response. Physiol.Zool. 71: 671-679. 

Purpose of Review: Registration Review

Date of Assessment: 4/20/12 – this review based on review from 4/06 (by Anita Pease) 

Brief Summary of Study Findings: 

Larval tiger salamanders (Ambystoma tigrinum) reared in the lab from eggs collected 

from a prairie wetland in North Dakota were exposed to atrazine at concentrations of 75 

ppb (81.83 ppb measured conc) and 250 ppb (299.7 ppb measured conc) in a static 

renewal test for 86 days to determine the effect on (1) plasma corticosterone and 

thyroxine concentrations, (2) larval size, and (3) days-to-stage at stages 2 and 4 of 

metamorphic climax. There were no exposure-related mortalities. Plasma thyroxine was 

elevated in both atrazine-exposed groups compared to the control, and plasma 

corticosterone was depressed in the 75 ppb treatment compared w/ both the control and 

the 250 ppb treatment group. Larvae exposed to 75 ppb atrazine reached stage 4 later, 

but at a size and weight comparable to the control group. Larvae in the 250 ppb 

treatment progressed to stage 4 at the same time, but at a smaller size and lower weight 

than larvae in the control group. 

Larvae exposed to atrazine responded with elevated thyroxine, causing curtailment of 

growth and acceleration of metamorphic progress. However, suppression of 

corticosterone may have moderated the effect of elevated thyroxine by slowing 

metamorphic progress and allowing additional growth between stages. The advantage is 

transformation to a larger size, with higher survival and fecundity prospects. Larvae 

subjected to 75 ppb atrazine took longer to undergo metamorphosis, indicating that they 

may spend more time in the wetland before metamorphosis (failure to transform before 

cold temps set in, thus making them vulnerable to anoxia in ice-covered wetlands). 

Larvae subjected to 250 ppb atrazine completed transformation at a smaller size 

(indicating potential lower survival and fecundity). 

Stress hormones (e.g., corticosterone) and thyroid hormones (e.g., thyroxine) promote 

differentiation associated with the onset of metamorphic climax. Plasma corticosterone 
138

and thyroxine concentrations rise in response to stressors in the environment as well as 

during metamorphoric climax. Corticosterone accelerates metamorphosis by promoting 

conversion of thyroxine to triiodothyronine in larvae in which circulating thyroid 

hormones are already elevated. Thyroxine not only promotes differentiation, but also 

retards growth in tiger salamanders. In larvae that have not reached the beginning of 

metamorphoic climax, elevated corticosterone concentrations have not been shown to 

influence metamorphic progress. 

Methods: Larvae (n = 34/treatment) were exposed to 3 concentrations of atrazine (0 

(acetone solvent), 75, and 250 ppb). Larvae were assigned to one of two groups: those 

that would be sampled at stage 2 (early) and those sampled at stage 4 (middle) of 

metamorphic climax. Larvae were tested individually in 7.6 L polyethylene containers 

covered w/screened lids, containing 4.0 L of test solution. Tests initiated when larvae 

SVL was 30 mm. Larvae were acclimated for a period of 2 days. Test solution renewed 

every second day by removing 1⁄2 of solution and adding equal volume of fresh solution. 

Larvae fed 3 red worm pieces daily, supplemented w/brine shrimp nauplii. Test temp = 

19-26 o

C. Dissolved oxygen reported as 20-70% during test. Water quality parameters 

were provided (pH = 8.1 + 0.1, cond at 25 o

C = 1045 + 11 umhos/cm, and hardness = 333 

+ 7 mg/L as CaCO3).

Description of Use in Document: INVALID. 

Rationale for Use: Atrazine measured in controls

Limitations of the Study: Atrazine was detected in the control sample at a measured 

concentration of 0.23 + 0.21 ppb. No raw data provided; lower dissolved oxygen 

reported and relatively wide ambient temperature range reported. No negative control 

tested and amount of solvent in treatment groups is unknown. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
139

3.34. Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin. 

2008. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Lenkowski, J.R., J. M. Reed, L. Deininger, 

and K. A. McLaughlin. 2008. Perturbation of organogenesis by the herbicide atrazine in 

the amphibian Xenopus laevis. Environ. Health Perspect. 116 (2): 223-230 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to examine the effects of atrazine exposure during organ 

morphogenesis in Xenopus laevis. 2,4 D and glyphosate were also tested to ensure 

observations were specific to atrazine and not general toxic effects; however this review 

focuses on atrazine. 

Methods 

Xenopus laevis embryos (≤15 tadpoles/10mL solution) (obtained from laboratory 

maintained adults) were exposed to atrazine concentrations of 10, 25 and 35 mg/L 

maintained at 18°C. All experiments were terminated at NF stage 47 (48 hours exposure) 

because all tests past this stage resulted in 100% mortality. A trial using both DMSO and 

ethanol was conducted to determine which solvent would be a better based on solvent 

toxicity. DMSO was chosen as the solvent used to prepare the atrazine concentrations as 

the study authors indicated it resulted in lower toxicity than ethanol. DMSO solvent 

concentrations ranged from 0.1 to 0.35%. Solutions were renewed every 24 hours and 

organisms monitored for mortality and malformations. At test termination, tadpoles were 

scored for circulatory malformations and then fixed and scored for edema and 

malformations of the heart, intestine and axis. Antibody localization, apoptosis in the 

brain and kidney was also evaluated. All exposures were performed two or three times 

with different tadpole broods. 

Results 

Mortalities were <5% in treatments and did not differ among groups. The study authors 

stated that malformations of the heart and visceral hemorrhaging and that atrazine 

significantly increased heart malformations across all treatments. Gut malformations and 

edema were also significant at all treatment groups. Axis malformations were significant 

at 25 and 35 mg/L. 
140

Description of Use in Document (QUAL, QUAN, INV):

Invalid 

Rationale for Use: 

The solvent concentration was greater than maximum recommended rate and DMSO not 

a recommended solvent. Also, reporting on certain test design elements were limited. 

Limitations of Study: 

This study appeared to be conducted similar to the ASTM FETAX assay. However, the 

use of DMSO at a solvent concentration of 0.1-0.35% is greater than the recommended 

rate for use in registration review (0.01-0.05%). Also DMSO is not a recommended 

solvent as it promotes movement across the membranes. The authors evaluated DMSO 

and ethanol for observed toxicity. While the incidence of observed toxicity was higher 

with ethanol, when combined with atrazine, the percent incidences using DMSO were 

always greater, unless 100% effect was observed in both solvents, with DMSO than 

ethanol suggesting that DMSO enhanced toxicity. In addition, there was limited reporting 

of the test design in terms of water quality, test vessel material, and analysis of test 

solutions. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
141

3.35. Lenkowski, J.R. and K. A. McLaughlin. 2010. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Lenkowski, J.R. and K. A. McLaughlin. 

2010. Acute atrazine exposure disrupts matrix metalloproteinases and retinoin signaling 

during organ morphogenesis in Xenopus laevis. J. App. Toxicol. 30: 582-589 (ECOTOX 

Ref No. 153851) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 2/27/12 

Summary of Study Findings: 

The objective of the study was to characterize tissue and molecular level effects of 

atrazine exposure during organ morphogenesis in X. laevis and to identify pathways by 

which acute atrazine exposure may affect developmental processes. 

Xenopus laevis tadpoles were exposed to atrazine concentrations of 0.035 – 35 ,g/L for 6-

48 hours. X. laevis eggs were collected as described previously in Lenkowski et al., 2008 

and embryos reared in 0.1x Marc’s modified Ringer’s (MMR) solution and embryos 

staged according to Nieuwkipp and Faber (1994). In results section study authors state 

that NF stage 41 was just prior to beginning experimental exposures. Atrazine stocks 

were prepared in dimethyl sulfoxide (DMSO) and diluted in 0.1xMMR. Final DMSO 

concentration was 0.35 %. Test solutions were renewed daily and were maintained at 

18°C. At test termination, tadpoles were euthanized and fixed or pooled for RNA 

transcript or zymographic analysis. Whole mount immunohistochemistry (IHC) was 

employed and muscle structure was examined using two muscle-specific antibodies 

(MF20 and 12/101). Tadpole cartilage was also examined. For reverse transcription, 20 

tadpoles pooled per treatment and all treatments performed in triplicate. Polymerase 

chain reaction (PCR) was used to examine 5 levels of matrix metalloproteinases MMPs 

that the authors state has described roles during X. laevis development (MMP2, 

MMP9TH, MMP11, MMP13, MMP18); three biological replicates were used. 

Zymography was used with 20 pooled tadpoles per treatment and protein concentrations 

were measured using the Bio-Rad Protein Assay and were repeated with four biological 
142

replicates. For the tadpole intestines, hybridization (in situ) was used and hybridization 

was visualized and representative photographs were taken. 

The study authors state that at 35 mg/L, acute atrazine exposure disrupted the structural 

integrity of differentiated cartilage and muscle and reduced and distorted cranial 

cartilage. By using RT-PCR, the study authors state that there was a significant effect of 

treatment across all MMPs examined with MMP9TH and MMP18 both increased 

expression with dose but only MMP18 significant at 3.5, 25 and 35 mg/L (Figure 

below). A RT-PCR analysis in a time course experiment was conducted and at 12 hours 

the authors reported an increase, although not significant, in MMP9TH, MMP11, and 

MMP18 at 35 mg/L; however MMP18 was significant at 6 and 48 hours for 35 mg/L. 

Zymography used to determine if atrazine altered MMP enzyme activity, and at 10 and 

35 mg/L, activity was decreased and increased compared to control; authors stated that is 

probably corresponded to MMP13. In the intestines, after 48 hours exposure at 10 and 35 

mg/L, gene (Xcyp26) expression significantly decreased and gene XRALDH2 also 

significantly decreased at 35 mg/L. The study authors state that based on this data, that 

atrazine does not inhibit development but rather causes alteration of already differentiated 

tissues and that the disruption may be through modulation of MMPs and the disruption of 

retinoic acid signaling. They also state that the data suggest that acute atrazine exposure 

causes the up-regulation, or prevents down-regulation, of specific MMPs. In addition, 

they state that acute atrazine exposure with changes in MMP transcript levels and protein 

activity affects MMP gene expression by at least two levels, misregulation of 

transcription and enzyme activity. While they state that the data was not presented, in 

atrazine exposed intestines, they have observed down-regulation of intestinal fatty acid 

binding protein in the intestines which they state is in response to thyroid hormone during 

metamorphosis. 
143

Figure 1 [Figure 2 in study report]. Atrazine exposure alters transcript levels and protein activity of MMPs. RT-PCR was 

used to measure the expression of MMP transcripts. Values shown are the average of three biological replicates. 

Expression of each MMP was divided by the internal control of a multiplex PCR reaction and normalized to matched untreated siblings. (A) Dose response of MMP expression levels. (B) MMP expression in response to 35mg l-1 atrazine after 

6, 12, 24 and 48 h of exposure. (C) MMP protein activity as determined by gelatin zymography fromwhole animal total 

protein lysates. Lanes from left to right are: (1)Molecular weight marker, (2) non-treated control, (3) 0.1% DMSO, (4) 

0.35% DMSO, (5) 10 mg l-1 atrazine, (6) 35 mg l-1 atrazine, (7) NF stage 60 tail protein, (8) NF stage 63 whole animal, (9) 

recombinant MMP9. Changes in gelatinase activity of atrazine-treated tadpoles are indicatedwith white arrows. 

Significance was determined by two-way ANOVA and Bonferroni corrected post-tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Description of Use in Document (QUAL, QUAN, INV): 

INVALID due to solvent choice and concentrations 

Rationale for Use: 

The solvent used in this study is not a preferred solvent as DMSO can promote 

movement across membranes and the solvent concentrations used were greater than the 

U.S. EPA guideline recommended concentrations. 
144

Limitations of Study: 

Test vessels used, loading, and water quality parameters were not reported. Measured 

concentrations of the test solutions were not reported. 

DMSO is not a preferred solvent as it promotes movement across membranes. The 

highest solvent concentration used in this study (0.35 %) is 7 or 35 times greater than the 

recommended solvent concentration for U.S. EPA OPPTS acute static (0.05%) or acute 

flow-through and chronic testing (0.01%), respectively. All solvent concentrations tested 

(0.035 to 0.35% DMSO) are greater than the recommended 0.01%. It is also not entirely 

clear why three different solvent concentrations were used and how they relate to the 

atrazine concentrations. 

The authors state that acute exposure to atrazine disrupted the structural integrity of 

differentiated cartilage and muscle and distorted cranial cartilage and were most apparent 

in the 35 mg/L group. This seems to suggest that these malformations were observed in 

other concentrations, however, the number and types of malformations observed per 

group are not reported. 

Primary Reviewer:Amy Blankinship, Chemist, Environmental Risk Branch 3, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively): 

Not applicable 

 
145

3.36. Lenkowski, J.R, G.Sanchez-Bravo, K.A. McLaughlin. 2010. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation Lenkowski, J.R, G.Sanchez-Bravo, K.A. 

McLaughlin. 2010. Low concentration of atrazine, glyphosate, 2,4-

dischlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus 

laevis organ morphogenesis. J. Environ. Sci. 22(9): 1305-1308. ECOTOX Ref No. 

153876) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/3/12 

Summary of Study Findings: 

The objective of the study was to evaluate the effects of atrazine, glyphosate, 2,4-D, and 

triadimefon on Xenopus laevis tadpoles during organ morphogenesis. This summary will 

focus on the studies conducted with atrazine. 

Methods 

X. laevis tadpoles were exposed to four concentrations of atrazine (purity not reported): 

0.35, 3.5, 10 and 35 mg/L along with two solvent concentrations (0.1 and 0.35% 

dimethyl sulfoxide (DMSO) and a negative control (0.1xMarc’s Modified Ringer’s 

solution (MMR). Tadpoles were exposed at NF stage 41 at 18oC for 48 hours with a 

static-renewal at 24 hours. Three replicates were used for each exposure and tadpole 

density per exposure was 13-18 tadpoles in 10mL of solution. Replicates were 

aggregated for analysis. Tadpoles were fixed, prepared and incubated overnight with a 

primary antibody known to identify X. laevis muscle sarcoplasmic reticulum. Antibody 

localization was visualized with a chromogenic reaction. 

Results 

 There was no mortality in the atrazine exposures. Intestinal malformations and edemas 

were significantly increased in the 3.5, 10 and 35 mg/L atrazine concentrations. Also, 

according to the study authors, the cranial muscles were reduced in the 35 mg/L atrazine 

concentration compared to the non-treated controls. The study authors state that the 

muscle degradation from atrazine exposure might be a direct effect of altered cell 

signaling that would breakdown the muscle tissue, an indirect effect due to the presence 

of edemas, or a toxic response. Also that the disruptions in hypaxial muscle could be a 

secondary effect of pesticide-induced edemas or the direct result of molecular changes 

induced by pesticide exposure, however, that was not addressed in the current study. 
146

They also state that axis malformations are common in atrazine-exposed tadpoles 

(Lenkowski et al., 2008). 

Description of Use in Document (QUAL, QUAN, INV): INVALID due to solvent 

choice and concentration 

Rationale for Use: 

Although the study does identify an increase in visual malformations in the intestines, 

cranial area and number of edemas, given that the atrazine concentrations were prepared 

with DMSO which is not a preferred solvent as it can promote movement across 

membranes. 

Limitations of Study: 

DMSO was used to prepare the atrazine concentrations. Solvent control concentrations 

of 0.1and 0.35% DMSO were reported as test concentrations. DMSO is not a preferred 

solvent as it can promote movement across membranes and potentially influence the test. 

Also, the concentration of the solvent should not exceed 0.5mL/L or 0.05% (ASTM 

2007); therefore, the solvent concentration was 2 and 7 times that recommended level. 

While not explicit in the study, the reviewer assumes that the solvent concentration in the 

atrazine treatment groups was either 0.1 or 0.35% DMSO. 

Environmental conditions other than temperature were not reported and although no 

mortality was reported, the reviewer cannot determine whether the tadpoles may have 

been stressed due to other environmental factors. Test vessels were not described. 

Loading in terms of mass tadpole/volume solution was not reported but rather in number 

of tadpoles/10mL. The use of 13-18 tadpoles/10mL solution may have increased the 

loading beyond the recommended 1 tadpole/L. 

While Figure 1 states that the DMSO controls were not significantly different than the 

non-treated controls, it is then presumed that the control referenced in Figure 1 is for the 

non-treated control given that the number of organisms is similar to the treatment groups 

and does not appear to represent a pooled control. 

 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

Reference: 

 
147

3.37. Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin. 

2008. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Lenkowski, J.R., J. M. Reed, L. Deininger, 

and K. A. McLaughlin. 2008. Perturbation of organogenesis by the herbicide atrazine in 

the amphibian Xenopus laevis. Environ. Health Perspect. 116 (2): 223-230 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 5/1/12 

Summary of Study Findings: 

The objective of the study was to examine the effects of atrazine exposure during organ 

morphogenesis in Xenopus laevis. 2,4 D and glyphosate were also tested to ensure 

observations were specific to atrazine and not general toxic effects; however this review 

focuses on atrazine. 

Methods 

Xenopus laevis embryos (≤15 tadpoles/10mL solution) (obtained from laboratory 

maintained adults) were exposed to atrazine concentrations of 10, 25 and 35 mg/L 

maintained at 18°C. All experiments were terminated at NF stage 47 (48 hours exposure) 

because all tests past this stage resulted in 100% mortality. A trial using both DMSO and 

ethanol was conducted to determine which solvent would be a better based on solvent 

toxicity. DMSO was chosen as the solvent used to prepare the atrazine concentrations as 

the study authors indicated it resulted in lower toxicity than ethanol. DMSO solvent 

concentrations ranged from 0.1 to 0.35%. Solutions were renewed every 24 hours and 

organisms monitored for mortality and malformations. At test termination, tadpoles were 

scored for circulatory malformations and then fixed and scored for edema and 

malformations of the heart, intestine and axis. Antibody localization, apoptosis in the 

brain and kidney was also evaluated. All exposures were performed two or three times 

with different tadpole broods. 

Results 

Mortalities were <5% in treatments and did not differ among groups. The study authors 

stated that malformations of the heart and visceral hemorrhaging and that atrazine 

significantly increased heart malformations across all treatments. Gut malformations and 

edema were also significant at all treatment groups. Axis malformations were significant 

at 25 and 35 mg/L. 
148

Description of Use in Document (QUAL, QUAN, INV):

Invalid 

Rationale for Use: 

The solvent concentration was greater than maximum recommended rate and DMSO not 

a recommended solvent. Also, reporting on certain test design elements were limited. 

Limitations of Study: 

This study appeared to be conducted similar to the ASTM FETAX assay. However, the 

use of DMSO at a solvent concentration of 0.1-0.35% is greater than the recommended 

rate for use in registration review (0.01-0.05%). Also DMSO is not a recommended 

solvent as it promotes movement across the membranes. The authors evaluated DMSO 

and ethanol for observed toxicity. While the incidence of observed toxicity was higher 

with ethanol, when combined with atrazine, the percent incidences using DMSO were 

always greater, unless 100% effect was observed in both solvents, with DMSO than 

ethanol suggesting that DMSO enhanced toxicity. In addition, there was limited reporting 

of the test design in terms of water quality, test vessel material, and analysis of test 

solutions. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
149

3.38. Oka, T., O. Tooi, N. Mitsui, M. Miyahara, Y. Ohnishi, M. Takase, A. 

Kashiwagi, T. Shinkai, N. Santo, T. Iguchi. 2008 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Oka, T., O. Tooi, N. Mitsui, M. Miyahara, 

Y. Ohnishi, M. Takase, A. Kashiwagi, T. Shinkai, N. Santo, T. Iguchi. 2008 Effect of 

atrzine on metamorphosis and sexual differentiation in Xenopus laevis. Aquat. Toxicol. 

87: 215-226. (ECOTOX Ref No. 103345) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/05/12 

Summary of Study Findings: 

The objective of the study was to examine the effects of atrazine on sex differentiation 

and P450 aromatase induction in wild-type X. laevis tadpoles (Experiment I) as well as 

the examining these effects in an all-ZZ male cohort of X. laevis tadpoles (Experiment 

II). 

Methods 

In experiment I, wild-type X. laevis tadpoles (from laboratory adult frogs; stage 49) were 

exposed to 0.1, 1, 10 and 100 μg/L atrazine (97.4%) as well as an ethanol solvent control 

(0.01%) until metamorphosis (stage 66). Two replicates (glass tanks filled with 10L 

solution) were used for each treatment and three replicates for the control with 30 

tadpoles per replicate; test solutions were changed three times weekly. Tadpoles were 

staged weekly and upon complete metamorphosis, total length and time to 

metamorphosis was measured. In experiment II, all ZZ male X. laevis tadpoles (stage 

49) were exposed to 0.1 and 1 μg/L atrazine, 0.27 μg/L E2, and 0.01% ethanol control; 

one replicate tank (30 tadpoles) was used for each treatment and frogs collected at 

metamorphosis. Stage 66 frogs were sexed by visual observation of gross morphology; 

after visual observation, the left gonad with kidney was removed for P450 aromatase 

determination and the right gonad was used for histological analysis from a subset of the 

exposed frogs. Liver samples were also taken for vitellogenin from a subset of frogs. In 

vitro vitellogenin assay using primary-cultured hepatocytes was also conducted but is not 

discussed in this review as it is an in vitro assay. 
150

Results 

Measured atrazine concentrations on exposure Day 0 were 0.2 , 1.7 , 14 and 120 μg/L or 

200, 170, 140, and 120% of nominal for the 0.1, 1, 10 and 100 μg/L group, respectively; 

after two days of exposure the concentrations were 0.1, 1.7, 17 and 150 μg/L for 0.1, 1, 

10, and 100 μg/L, respectively. In experiment I, survival was >90% in the control and 1 

and 10 μg/L groups, but lower survival was reported for one of the two tanks in the 0.1 

and 100 μg/L, 73.3 and 26.7%, respectively; as such, these two tanks were not used in 

subsequent analyses. Sex ratio was significantly different for the 10 and 100 μg/L 

atrazine groups compared to the control; no hermaphroditism was observed (Table 1). 

According to the study authors, evaluation of sex by gross morphology was corroborated 

by histological analysis. Time to metamorphosis was similar among treatment groups 

and there did not appear to be an acceleration or inhibition of development [statistical 

results/p value not reported].

In experiment II, survival was >96%. According to the authors, there was no evidence of 

sex-reversal in all male cohorts exposed to atrazine but was observed in the E2 group 

(Table 2). IN the control and 1-100 μg/L groups, expression of P450 aromatase mRNA 

was higher in females than males. In both experiments, gonad P450 aromatase mRNA 

induction was not observed in frogs exposed to atrazine but was induced in the all male 

frogs exposed to E2. 

Description of Use in Document (QUAL, QUAN, INV): 

INVALID for metamorphosis as data not presented and QUAL for other endpoints 
151

Rationale for Use: 

The paper provides some information about the effects of atrazine on sexual development 

in X. laevis. However, the lack of a negative control as well as the variability in survival 

for two of the atrazine treatments and the measurement of estrogenic activity in the food 

for the adult X. laevis frogs used to collect larvae raise uncertainties in the conclusions of 

this study. 

Limitations of Study: 

This study was conducted without a negative control. Therefore, potential effects from 

the solvent could not be evaluated. 

The food source for the adult frogs used as the source of eggs for the study was reported 

to show slight estrogenic activity in the yeast screening assays of which the authors 

suggested may be a phytoestrogen. It is not known how this contaminate may have 

influenced the study. The food source for the tadpoles during the study was reported to 

be free of estrogenic activity. In addition, other than chlorine, characteristics of the water 

source were not reported. 

Variable mortality in replicate tanks was reported for two of the treatment groups, in 

which survival was 73 and 27% in one of the two replicate tanks for the 0.1 and 100 μg/L 

groups, respectively. A source of this variability was not reported; therefore there is 

uncertainty as to what other possible stressors or changes in environmental conditions the 

tadpoles may have encountered. 

The use of one replicate in Experiment II did not allow for evaluation of variability 

within a treatment group. Statistical results for metamorphosis not reported. 

Total number of animals with ovaries, testes, and hermaphroditism and survival per 

treatment group are reported. Variability between replicates not reported. 

The reported measured concentrations of atrazine where substantially greater than 

nominal (ranged 120-200% of nominal). The study reported a Day 0 and Day 2 analysis. 

It is unclear whether these are the mean concentrations from the renewals or if they 

represent only one Day 0 or 2 sampling period. Results for the control are not reported 

(unclear if measured). 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
152

3.39. Olivier, H. M. and R. R. Moon. 2010. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Olivier, H. M. and R. R. Moon. 2010. The 

effects of atrazine on spotted salamander embryos and their symbiotic alga. 

Ecotoxicology 19: 654 – 661. (ECOTOX Ref No. 153629) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 10/29/11 

Summary of Study Findings: 

Egg masses (approximately 2- 3 days after laying) of spotted salamanders (Ambystoma 

maculatum) collected from pond in Evangeline Parish, Louisiana. Each egg mass, 

consisting of 26 - 127 eggs, constituted the treatment unit; 20 egg masses assigned to 

each treatment group consisting of the atrazine formulated product AAtrex 4Lr [42.6%; 4 

lbs ai/gal] at 0, 50, 100, 200, and 400 ug/L. During the 76-day exposure period [based on 

Figure 3] exposure solutions were renewed on exposure day 21. Individual egg masses 

were enumerated (number of eggs per egg mass) and were maintained in deionized water 

within 0.75L plastic containers. Aggregate (reached by 90% of the embryos) 

developmental stage (based on the methods or Harrison 1969), number of dead eggs and 

the presence/absence of algae were recorded. At the start of the study, Harrison 

development stages ranged between 10 – 17. Algal growth determined by scoring the 

intensity of green coloration against coded [paint] color palettes; this was in turn used as 

“reasonable indicator of the potential for oxygen production” since photosynthesis 

requires the green pigment chlorophyll. Hatching success for each egg mass determined 

by dividing the number of hatchlings by the number of eggs originally in the mass. Final 

sample size used in analysis for hatching success summarized in Table 1.

Highest developmental stage reached for dead or hatched embryos also determined. 

Approximately 2 weeks after hatching began, when no more living embryos in the egg 

masses, all animals were euthanized. 

 
153

Table 1. Summary of egg mass sample sizes used for hatching success evaluation; 

original number of egg masses N=20.

Sample 

Size

Atrazine Concentration ug/L 

0 50 100 200 400 

16 12 14 18 20 

All atrazine concentrations resulted in complete bleaching which was interpreted by the 

study authors as complete mortality of the algae and “probably led to severely reduced 

oxygen production.” Control egg masses displayed bright green algae “indicating high 

algal growth and a high potential for photosynthesis and oxygen production. 

“Pathologies” (hemorrhages, spinal curvatures, malformed fins) developed in most of the 

atrazine-treated eggs immediately following the malfunction of the climate control units; 

these pathologies disappeared within 5 days in all but one egg mass. Timing and 

mortality of egg masses are reported to be directly related to atrazine concentration. 

Based on the figures provided, the study appears to have been conducted for 76 days. 

Egg mass mortality was 90% by day 68 in the 200 ug/L treatment and 100% by day 38 in 

the 400 ug/L treatment. Extrapolating off of Figure 3 of the study, egg mass mortality in 

0, 50, 100, 200 and 400 ug/L treatments was 0, 5, 35, 90 and 100%, respectively. 

Again extrapolating from report figures, hatching success in the 0, 50, 100, 200 and 400 

ug/L treatments was 97%, 36%, 14%, 2% and 0%, respectively. 

As with hatching success, development stage reached was significantly (p<0.05) 

inversely related to atrazine concentration. Developmental stage did not differ 

significantly between 0 and 50 or between 50 and 100 ug/L. In controls the highest 

development stages reached were between 42 – 45. In the 100 ug/L treatment 

development max was between 36 – 42; in the 200 ug/L, it was 31 – 42; the highest 

developmental stage in the 400 ug/L group was 25 – 36. 

The authors conclude that atrazine exposure affected the spotted salamander both directly 

and indirectly by causing pathologies and mortality in embryos and indirectly by 

eliminating symbiotic algae. 

Description of Use in Document (QUAL, QUAN, INV): 

Qualitative 

Rationale for Use: 

Study has a number of confounding effects; although controls appear to have functioned 

relatively well, the data strongly suggest that the failure of the climate control unit had a 

marked effect on other treatment groups. The study does not provide any water quality 

information and a number of assumptions are made regarding the condition of algae and 

potential photosynthesis/oxygen depletion. While effects on [apparent] chlorophyll and 

mortality are documented, the use of a formulated product that is not labeled for use in 

water makes it difficult to assign causality directly to atrazine. 

Limitations of Study: 
154

AAtrex® 4L label contains 42.6% active ingredient; however, the reviewer was not able 

to locate the AAtrex 4Lr label specifically. It’s not clear from the publication whether 

exposure concentrations were in terms of formulated product or active ingredient. The 

4L product is restricted use and prohibits direct applications to water or within 50 ft of 

surface water. 

 Wild-caught egg masses were used in the study; prior exposure history is unknown. 

Authors do not state whether the egg masses were uniform in size and what the 

dimensions of the egg masses were. It is uncertain whether each egg mass contained 

roughly 5,628 embryos or whether the entire study utilized this number of embryos. 

Authors assume synchronous development of the egg mass and based their understanding 

of the entire egg mass on the performance of the outermost embryos. The fact that 

Harrison developmental stages at the start of the study ranged between 10 – 17 suggests 

that the embryos were not in synchronous development. 

Use of deionized water; authors claim that a retrospective analysis of the results indicated 

that the use of deionized water did not impair the successful hatching, development and 

larval survival of controls. Deionized water was selected since “it would not harm the 

eggs or larvae and because it would not introduce any new algae into the solutions.” 

The text states that the study was initiatied on February 1 and that the dilution water was 

replenished on Feb 22; this implies a 21-day renewal period. However, Figure 3 of the 

study indicates that water was replenished at 28 days. The 21- or 28-day renewal period 

may have been excessively long; however, without water quality information it is 

difficult to determine. Study was conducted in plastic containers which may have had 

plasticizers which could have impacted animal development. 

By Day 19 of the study, climate control units failed and water temperatures increased 

from 3o

C to 10oC. 

Authors relied on an aggregate color assessment to determine status of algae; they 

equated the visual absence of color to algae death [and oxygen depletion] and did not 

verify whether this assumption was justified and whether oxygen concentrations had 

actually been depleted. 

Authors acknowledge that the use of formulated endproduct prevented them from 

assigning any effects observed in the study exclusively to atrazine. 

The authors acknowledge that it was difficult to accurately determine the number of 

embryos inside the large three dimensional egg masses and therefore there were “some” 

discrepancies between the initial count and final counts/unhatched eggs and they 

excluded from analysis those egg masses for which discrepancies were >10%. 

It is unclear from the study results whether mortality of the egg masses refers to the entire 

egg mass or individual eggs within the egg mass. It is note worthy that the reported 

“pathologies” were initiated immediately after the failure of the climate control units, that 

the pathologies were transitory (lasting roughly 5 days for all but one egg mass) and the 

mortality started abruptly at day 30 corresponding closely to when the treatment water 

was replenished. 

Primary Reviewer: 
155

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
156

3.40. Orton F., I. Lutz, W. Kloas, and E.J. Routledge. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Orton F., I. Lutz, W. Kloas, and E.J. 

Routledge. 2009. Endocrine disrupting effects of herbicides and pentachlorophenol: in 

vitro and in vivo evidence. Environ. Sci. Technol. 43: 2144-2150. (Ecotox 11711). 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/30/12 

Summary of Study Findings: 

This paper describes in vitro assays (recombinant yeast screen to detect receptor mediated 

anti estrogenic and androgenic activity and cultured Xenopus oocytes to measure effects 

on ovulatory response and ovarian steroidogenesis in Xenopus laevis. An in vivo study 

was conducted with pentachlorophenol. As atrazine was analyzed in vitro assays and not 

in in vivo assays, this study will not be used in the risk assessment 

Description of Use in Document (QUAL, QUAN, INV): 

Not applicable 

Rationale for Use: 

Not applicable 

Limitations of Study: 

Not applicable 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 Not applicable 
157

3.41. Relyea, R. A. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Record Number and Citation: 

Relyea, R. A. 2009. A cocktail of contaminants: how mixtures of pesticides at low 

concentrations affect aquatic communities. Oecologia 159(2): 363-376. (MRID 

48261129) 

Purpose of Review: 

Determine potential effects of atrazine on an aquatic community. Endpoints from this 

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant 

communities and associated determination of a Level of Concern (LOC). 

Date of Review: 

October 15, 2009 

Purpose of Study: 

This study examines the effect of five insecticides (malathion, carbaryl, chlorpyrifos, 

diazinon, and endosulfan) and five herbicides (glyphosate, atrazine, acetochlor, 

metolachlor and 2,4-D) at low concentrations on outdoor mesocosms composed of 

zooplankton, phytoplankton, periphyton, and larval amphibians (gray tree frogs, Hyla 

versicolor and leopard frogs, Rana pipiens). The pesticides were studied alone, as a 

mixture of the five herbicides, as a mixture of the five insecticides, and as a mixture of all 

ten herbicides and insecticides. 

Summary of Study Findings: 

The focus of the summary is on the mesocosms treated with atrazine alone. 

Methods: 

The experiment was conducted at the University of Pittsburgh’s Pymatuning Laboratory 

of Ecology in northwestern Pennsylvania (USA). The experiment used a completely 

randomized design consisting of 15 treatments with 4 replicates each for a total of 59 

experimental tanks (the solvent control consisted of only 3 replicates). The treatments 
158

were as follows: a negative control (water), a vehicle control (ethanol), each of the five 

insecticides applied separately (malathion, carbaryl, chlorpyrifos, diazinon, and 

endosulfan), each of the five herbicides applied separately (glyphosate, atrazine, 

acetochlor, metolachlor, and 2,4-D), a mixture of all five herbicides, a mixture of all five 

insecticides, and a mixture of all ten pesticides. 

The mesocosms were 1,300-L cattle watering tanks. The tanks were filled (on May 15-

19, 2006) with about 1000 L of well water (pH 8). On June 7th, the tanks were 

supplemented with a mixture of water from nearby ponds which contained zooplankton, 

phytoplankton, and periphyton. The following day, each tank was further supplemented 

with 300 g of leaf litter (primarily Quercus spp.) and 25 g of commercial rabbit chow 

(source of algal nutrients and additional surface for algal growth). On June 16th, the 

periphyton samplers were added to each tank in the form of two unglazed clay tiles (10 × 

10 cm, oriented vertically). After 18 days (June 25th), the tadpoles were added. The 

tadpoles were collected for the experiment as newly oviposited egg masses and hatched 

in 200-L wading pools where they were fed rabbit chow ad libitum until use in the 

experiment. The time at which each species was oviposited differed (ten masses of 

leopard frogs, collected on March 31st; 23 masses of gray tree frogs, collected on May 

14th and 17th), thus the two species were of different ages when added to the experiment. 

Added to each tank were 20 tadpoles of each species (H. versicolor, and R. pipiens) 

chosen from a mixture of all egg masses (initial mass ± SE: gray tree frogs, 77 ± 4 mg; 

leopard frog, 134 ± 12 mg). The density of tadpoles in the experimental tanks was 9/m2

for each species, which was within natural densities for the two species. 

Two days after tadpole addition, the tanks were exposed to atrazine (and other 

treatments) via a single dose of technical grade chemical (chemical purity, 98.1%) at a 

nominal concentration of 10 μg/L (p.p.b.) using ethanol as the vehicle. Ethanol 

concentration was 0.003% in the solvent control tanks and the tanks receiving a mixture 

of all ten pesticides (ethanol concentration in the atrazine-only treatment was not stated). 

Control tanks without ethanol were also included. Atrazine was added to the tanks and 

the surface water was mixed with a 500-mL container. One hour later, water samples 

were collected midway in the water column; samples from the four replicate tanks were 

pooled and analyzed for atrazine by HPLC. The measured concentration of atrazine was 

6.4 μg/L (p.p.b.). The control tanks were not analyzed for any of the ten pesticides. The 

author states that the well water used for filling the tanks did not have detectable 

concentrations of any of the pesticides. However, it was not stated if the pond water 

(biota source) added to the tanks was analyzed for any of the pesticides. 

The temperature, pH, and dissolved oxygen in each tank were measured using a digital 

water meter on days 10 and 35 of the experiment. 

On days 16 and 36, the zooplankton was sampled midway in the water column at the four 

cardinal directions and the center of the tank using a 0.2-L tube sampler. The five 

samples from each tank were combined, filtered (62-μm Nitex screen), and then 

preserved in 70% ethanol. The zooplankton was classified to species. Twelve taxonomic 
159

groups of cladocerans and copepods were detected. The assemblage was dominated 

(91%) by four species: two species of cladocerans (Daphnia pulex and Ceriodaphnia sp.) 

and two species of copepods (Skistodiaptomus oregonensis and Leptodiaptomus 

minutus). 

On days 16 and 35, phytoplankton was sampled via collecting 500 mL from the middle of 

the water column in each tank. The samples were vacuum filtered through a Whatman 

GF/C filter. Chlorophyll a concentrations were determined by fluorometry after the 

filtered samples were prepared following the protocols of Arar and Collins 1997 

(including acidification). 

On days 25 and 36, the periphyton was sampled via scrubbing one side of the unglazed 

clay tile into a tub of filtered water. After filtration of the slurry (Whatman GF/C filters, 

pre-dried at 80°C and weighed) the filters were dried (80°C for 24 hours) and reweighed 

again to determine the biomass of the periphyton. 

Metamorphs were removed from the tanks when they had at least one emerged forelimb 

(stage 42); the gray tree frogs emerged first due to their inherently shorter time to 

metamorphosis. Daily searches of each tank were conducted starting day 21 (July 17) of 

the experiment when the first metamorph was observed through day 57 (August 22nd, last 

day of experiment). After removal, the metamorphs were held in one liter laboratory tubs 

containing moist sphagnum moss until tail resorption was complete (stage 46). The 

number of days from the beginning of the experiment to stage 46 was defined as time to 

metamorphosis. Each animal was euthanized in 2% MS-222, preserved, and weighed. 

The response variables for the amphibians were percent survival, mean time to 

metamorphosis, and mean mass at metamorphosis. 

After most of the metamorphs of both species had emerged (determined later to be 97.3% 

of all live tadpoles), approximately 120 L of water was removed from each tank daily 

from day 50 to and including day 57 in order to simulate pond drying (and accelerate 

metamorphosis). On day 57, the tanks were classified as “dry ponds”. The remaining 

amphibians were classified as dead, non-emergent (due to slow growth and 

development), or a successful metamorph (possessing at least one emerged forelimb; 

these were treated as described above). 

The response variables were analyzed using multivariate ANOVA (MANOVA). The 

MANOVA was composed of the second measurements of all abiotic conditions, 

cladoceran and copepod abundance, phytoplankton abundance (chlorophyll a), 

periphyton biomass, the survival of gray tree frogs and leopard frogs, and size at and time 

to metamorphosis of gray tree frogs. The metamorphosis data could not be collect for the 

leopard frogs in two of the pesticide treatments due to nearly 100% mortality, so the 

leopard frog history responses were analyzed in a separate MANOVA. Each univariate 

response variable was examined using either an ANOVA (for responses that were 

measured at the end of the experiment: tree frog mass, tree frog time to metamorphosis, 
160

tree frog survival and leopard frog survival) or a repeated-measures ANOVA ( for 

responses measured twice during the experiment: temperature, dissolved oxygen, pH, the 

abundance of cladocerans (D. pulex and Ceriodaphnia sp.), the abundance of copepods 

(S. oregonesis and L. minutus), phytoplankton (chlorophyll a) and periphyton biomass. 

The data was log-transformed when necessary. The zooplankton data was ranked prior to 

analysis since it had heteroscedastic errors. The multivariate analyses controlled the 

experiment-wise error rate at α = 0.05. To maximize the power of the analyses, post hoc 

comparisons were conducted using Fisher’s LSD test which preserves the comparisonwise error rate at α = 0.05. 

Results: 

The MANOVA on all final response variables (excluding the two life history traits of 

leopard frogs) revealed a significant effect of the treatments (Wilks’ λ, F182,313 = 2.5, P < 

0.001); therefore, the ANOVA analyses were conduct for each response. These analyses 

were not specific to atrazine-only treatments and will not be discussed further. 

 Amphibians 

For leopard frog tadpoles (R. pipiens), there was not a significant difference (P > 0.05 

using Fisher’s LSD test) between the atrazine treatment and the no-pesticide treatments 

(i.e. negative and ethanol control tanks) regarding survival to metamorphosis, mass at 

metamorphosis, and time to metamorphosis (Figure 1). For gray tree frog tadpoles (H. 

versicolor), there was not a significant difference between the atrazine treatment and the 

no-pesticide treatments (P > 0.05 using Fisher’s LSD test) regarding survival to 

metamorphosis and time to metamorphosis (Figure 2). However, for mass at 

metamorphosis, gray tree frogs were larger in tanks treated with atrazine compared to 

those in the negative and ethanol control tanks (P = 0.045). 
161

Figure 1. Changes in the (a) survival, (b) mass at metamorphosis, and (c) time to metamorphosis of 

leopard frog tadpoles (Rana pipiens) in outdoor mesocosms exposed to no pesticides (control), solvent 

only (ethanol), five separate insecticides (I) (carbaryl, malathion, chlorpyrifos, diazinon, and 

endosulfan), five separate herbicides (H) (acetochlor, metolachlor, glyphosate, 2,4-D, and atrazine), a 

mixture of the five insecticides, a mixture of the five herbicides, and a mixture of all ten pesticides. In 

two of the treatments, there were no life history data (ND) due to a lack of surviving animals. Data 

are mean ± 1 SE. Asterisks indicate treatments that are significantly different from the no-pesticide 

treatment (P < 0.05 using Fisher’s LSD test). Figure 1 from Relyea 2009. 

 
162

Figure 2. Changes in the (a) survival, (b) mass at metamorphosis, and (c) time to metamorphosis of 

gray tree frog tadpoles (Hyla versicolor) in outdoor mesocosms exposed to no pesticides (control), 

solvent only (ethanol), five separate insecticides, five separate herbicides, and a mixture of all ten 

pesticides. Data are mean ± 1 SE. Asterisks indicate treatments that are significantly different from 

the no-pesticide treatment (P < 0.05 using Fisher’s LSD test). For abbreviations, see Figure 1. Figure 

2 from Relyea 2009. 

 Zooplankton

The abundance of the four species which dominated (91%) the zooplankton was 

quantified: two species of cladocerans (D. pulex and Ceriodaphnia sp.) and two species 

of copepods (S. oregonensis and L. minutus). There was no effect from the atrazine 

treatment (P > 0.05 using Fisher’s LSD test) relative to the control regarding the 

abundance of these species (Figure 3). 
163

Figure 3. Changes in the abundance of copepods [(a) Leptodiaptomus minutus and (b) 

Skistodiaptomus oregonensis] and cladocerans [(c) Ceriodaphnia sp. and (d) Daphnia pulex] in 

outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), five separate 

insecticides, five separate herbicides, and a mixture of all ten pesticides. Data are mean ± 1 SE, 

averaged over two sample periods (day 16 and 36), on a log scale. Asterisks indicate treatments that 

are significantly different from the no-pesticide treatment (P < 0.05 using Fisher’s LSD test). For 

abbreviations, see Figure 1. Figure 3 from Relyea 2009. 

 Phytoplankton and Periphyton 

For chlorophyll a (phytoplankton) measured on days 16 and 35, there was not a 

significant difference between the atrazine treatment and the no-pesticide treatments (P > 

0.05 using Fisher’s LSD test). For periphyton biomass measured on days 25 and 36, 

there was not a significant difference between the atrazine treatment and the no-pesticide 

treatment (P > 0.05 using Fisher’s LSD test). Phytoplankton and periphyton results are 

illustrated in Figure 4. 
164

Figure 4. The abundance of (a) phytoplankton (measured as chlorophyll a concentration) and (b) 

periphyton (measured as dry biomass on a clay tile) over time in outdoor mesocosms exposed to no 

pesticides (control), solvent only (ethanol), five separate insecticides, five separate herbicides, and a 

mixture of all ten pesticides. Data are mean ± 1 SE on a log scale. Within each sample date, asterisks 

indicate treatments that are significantly different from the no-pesticide treatment (P < 0.05 using 

Fisher’s LSD test). For abbreviations, see Figure 1. Figure 4 from Relyea 2009. 

 Abiotic Effects 

Tank water pH and dissolved oxygen were measured on days 10 and 35. There was not a 

significant difference between the atrazine treatment and the negative control (P > 0.05 

using Fisher’s LSD test) for either pH or dissolved oxygen on either date (Figure 5). The 

ethanol control differed from the negative control on day 10 for both pH and dissolved 

oxygen but was similar to the negative control on day 35. 
165

Figure 5. Changes in (a) pH and (b) dissolved oxygen over time in outdoor mesocosms exposed to no 

pesticides (control), solvent only (ethanol), five separate insecticides, five separate herbicides, and a 

mixture of all ten pesticides. Data are mean ± 1 SE. Within each sample date, asterisks indicate 

treatments that are significantly different from the no-pesticide treatment (P < 0.05 using Fisher’s 

LSD test). For abbreviations, see Figure 1. Figure 5 from Relyea 2009. 

 

 Overall Results 

Overall, based on the results of the study, the study author concluded that atrazine had no 

significant effect (P > 0.05 using Fisher’s LSD test) on the biomass of any of the trophic 

groups monitored in this outdoor mesocosm experiment. The only significant effect of 

atrazine treatment which was observed was the increase in mass at metamorphosis for 

gray tree frog tadpoles (P = 0.045). The study author reported that the mechanism for 

this effect was unclear. The overall effects of the controls and atrazine treatments on 

biomass of trophic groups in the food web are qualitatively illustrated in Figure 6. 
166

Figure 6. Changes in biomass of trophic groups in the food web as a result of exposure to (a) no 

pesticide (control), (b) solvent control (ethanol) and (i) atrazine. The size of the circle and font for 

each trophic group indicates the qualitative change in the biomass of that trophic group. R Basal 

resources for the two types of algae. Figure 6 from Relyea 2009. 
167

Data Summary Table: 

Effect 

Magnitude of 

Effect relative 

to controls 

Nominal 

Atrazine 

Concentration 

(μg/L)1 

Day of 

Study Effect 

Occurred 

Time 

Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Chlorophyll a – 

phytoplankton No effect 6.4 μg/L Days 16 and 

35 Not applicable Not applicable

P > 0.05 

Biomass – 

periphyton No effect 6.4 μg/L Days 25 and 

36 Not applicable Not applicable

P > 0.05 

Dissolved 

oxygen No effect 6.4 μg/L Days 10 and 

35 Not applicable Not applicable 

P > 0.05 

Survival to 

metamorphosis

– R. pipiens No effect 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Not applicable Not 

applicable 

P > 0.05 

Mass at 

metamorphosis

– R. pipiens No effect 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Not applicable Not 

applicable 

P > 0.05 

Time to 

metamorphosis

– R. pipiens No effect 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Not applicable Not 

applicable 

P > 0.05 

Survival to 

metamorphosis

– H. versicolor No effect 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Not applicable Not 

applicable 

P > 0.05 

Mass at 

metamorphosis

– H. versicolor ~23% increase 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Could not be 

determined. 

Could not be 

determined. 

P = 0.045 

Time to 

metamorphosis

– H. versicolor No effect 6.4 μg/L 

Varied, 

depending 

on tadpole 

emergence 

Not applicable Not 

applicable 

P > 0.05 

L. minutus 

abundance No effect 6.4 μg/L Days 16 and 

362 Not applicable Not 

applicable 

P > 0.05 

S. oregonensis 

abundance No effect 6.4 μg/L Days 16 and 

362 Not applicable Not 

applicable 

P > 0.05 

Ceriodaphnia

sp. abundance No effect 6.4 μg/L Days 16 and 

362 Not applicable Not 

applicable 

P > 0.05 

D. pulex 

abundance No effect 6.4 μg/L Days 16 and 

362 Not applicable Not 

applicable 

P > 0.05 

1

 Nominal concentration was 10 μg/L. Actual concentration (6.4 μg/L) was measured 1 hour after 

application. 

2 Data analyzed was the average zooplankton abundance over two sampling dates (days 16 and 36). 
168

Reviewer Comments: 

No measurements were made to determine if atrazine and the other nine pesticides were 

present in the control tanks. The author states that the well water used for filling the 

tanks did not have detectable concentrations of any of the pesticides used in the study, 

although the data or time at which it was analyzed was not reported. In addition, it was 

not stated if there was chemical analysis for any of the pesticides in the pond water which 

was added to the tanks as the source of the planktonic biota. 

The author reports that the concentration of ethanol was 0.003% in the solvent control 

tanks and the tanks receiving a mixture of all ten pesticides but does not report if the 

concentration of ethanol in the atrazine-only treatment was also 0.003%. 

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the weight of evidence regarding the 

potential for effects of atrazine on aquatic plant communities.

Limitations of Study: The results have good applicability to mesocosms or natural 

aquatic systems because the experimental mesocosms were outdoors during testing and 

contained phytoplankton, periphyton, zooplankton, and two species of amphibians. 

Actual pesticide (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. 

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc 

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA 

 
169

3.42. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. 

McCarthy, J.H. Niedzwiecki, T. Sager, A. Sih, and B.D. Palmer. 2003 

OPEN LITERATURE REVIEW 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 71723 

Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. 

Niedzwiecki, T. Sager, A. Sih, and B.D. Palmer. 2003. Lethal and Sublethal Effects of 

Atrazine, Carbaryl, Endosulfan, and Octylphenol on the Streamside Salamander 

(Ambystoma barbouri). Environmental Toxicology and Chemistry, Vol. 22, No. 10. p. 

2385-2392. ECOTOX 71723 

Purpose of Review: Registration Review

Date of Assessment: 4/20/12 (this review based on review done on 4/06) 

Brief Summary of Study Findings: 

Streamside salamander embryos and larvae were exposed to atrazine (80% ai) for 37 days 

at concentrations of 4, 40, and 400 ppb in the presence and absence of food (carbaryl, 

endosulfan, and octylphenol were also examined). No effect on embryo or larval 

survival, hatching, or growth or development (i.e., mass, SVL, and limb deformities) 

rates were observed at any of the test concentrations. Systematically tapping tanks 

caused a greater activity in larvae exposed to 400 ppb atrazine, suggesting underlying 

nervous system malfunction according to the study author. Hunger stimulated a decrease 

in refuge use and an increase in activity (based on regression; no significant difference 

detected at any level); however this response was least pronounced in the larvae exposed 

to atrazine at 400 ppb, possibly because these larvae were the most lethargic. 

Methods: 

12 egg clutches collected from Fossil Creek, KY. 4 rep bowls per treatment (10 

animals/bowl = total 40 animals per treatment. Atrazine treatments contained solvent 

DMSO; solvent control contained DMSO and acetone (acetone used as solvent for other 

chemicals tested). Water was changed every other day. During water changes, number 

of live embryos and larvae were recorded, larvae were fed live blackworms ad libitum, 

and bowls were randomly shuffled to control for location effects. Exposure to atrazine 

began 2 days after egg collection and lasted for 37 days, but survival was tracked for 53 

d. Larval behavior was recorded in presence and absence of food; on exp day 16 and 28, 

blackworms were removed and number of larvae under refuge and moving in 30 s was 

recorded. Tank tapping occurred via a spring-loaded mousetrap and number of larvae 

moving after disturbance (referred to as reflex activity) was recorded. 
170

Description of Use in Document: Qualitatively to discuss NOAEC (400 ppb); no 

effects to survival, hatching, or growth at highest test concentration. 

Rationale for Use: Information on the toxicity of atrazine to streamside salamanders 

Limitations of the Study: Solvent control contained both DMSO and acetone, whereas 

the atrazine treatment groups contained DMSO only. DMSO is not an acceptable solvent 

because it accelerates the movement of a chemical across cell membranes. No raw data 

provided. The amount of solvent added to the treatment groups is higher than the 

recommended maximum solvent concentration: 1.25mL stock in 4L water ((1.25/4000) 

*100= 0.03%; in addition the concentration in the solvent control appeared to be twice 

that as is seems that 1.25mL of both DMSO and acetone were added to the solvent 

control. 

The study did not report is the controls were analyzed for the presence of the test 

chemicals. The organisms were field collected so prior exposure history is unknown and 

the authors state that it is likely that they were exposed to the test chemicals because they 

were collected from an area influenced by agricultural activity (chemical measurement 

for the four test chemicals at the collection site not available). Limited water quality 

parameters were reported (only temperature) and the loading was greater than the 

recommended 1 tadpole (larvae) per liter solution. Growth parameters were measured at 

Day 50 and analyzed. The stage of the organisms at Day 50 were not reported but if there 

was variability in the stages, it could potentially influence the growth analysis as mass 

and length are correlated with development. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
171

3.43. Rohr, J.R. and B.D. Palmer. 2005 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 81664 

Rohr, J.R. and B.D. Palmer. 2005. Aquatic herbicide exposure increases salamander 

desiccation risk eight months later in a terrestrial environment. Environ. Toxicol. Chem. 

24(5): 1253-1258. 

Purpose of Review: Registration Review 

 

Date of Assessment: 5/1/12 

Brief Summary of Study Findings: 

The Ambystoma barbouri salamander metamorphs used in this study were obtained from 

a previous experiment in which embryos were exposed to 0, 4, 40 or 400 μg/L atrazine 

exposures till metamorphosis (Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM, 

Niedzwiecki JH, Sager T, Sih A, Palmer BD. 2004. Multiple stressors and salamanders: Effects of an 

herbicide, food limitation, and hydroperiod. Ecol Appl 14:1028–1040). After metamorphosis, the 

organisms were maintained in moist terraria at 20°C with a 12:12 photoperiod. After a

mean of 130 days after atrazine exposure, metamorphs from each treatment were 

distributed singularly or in groups of three into petri dishes at a room temperature of 

26°C (relevant mean summer soil temperature according to author). Petri dishes lined 

with filter paper and weighed before and after adding metamorphs. Every hour for 5 

hours recorded mass of metamorphs and observed water-conserving behaviors (moving 

and pressed against dish and number of organisms huddled (when dished contained 3 

metamorphs)). Test was repeated one week later using different animals. The same 

method description above was used to retest the same animals a mean of 237 and 239 

days after atrazine exposure but only one animal was added to each dish. 

No salamanders died during the trials. There was no difference between the earlier trial 

and the later one for behavior or mass loss and subsequent results are based on both trials. 

According to the authors, the salamanders exposed to 40 and 400 μg/L exhibited greater 

activity and fewer water-conserving behaviors and accelerated water loss compared to the 

control for both the four and eight months post atrazine exposure (see figures below 

reprinted from study). 
172
173

Description of Use in Document: Qualitative 

 

Rationale for Use: The study provides information about the behavior of the 

salamanders post metamorphosis, however, there are uncertainties regarding the 

organisms conditions from the other study. 

Limitations of the Study: 

Limitations that were described in Rohr et al. 2004 (E81748) would apply here given that 

the organisms were from the same study. In that study, a white film was observed on 

dead embryo and it was unknown if fungus caused or followed mortality. No negative 

control (without atrazine or solvent) was included in test. Also, larval survival 

differences between years, and although pH, temp, and DO were adequate, nitrates and 

ammonia not reported and given the higher loading and with weekly renewal, there is 

uncertainty in whether ammonia levels were within acceptable limits. 

In addition, DMSO is not a recommended solvent. Organisms field collected and 

therefore prior exposure unknown. Study authors have previously reported that 

organisms likely exposed due to agricultural practices and reported an atrazine 

concentration of 33.9 ppb in a similar sized stream in same state as collection site. 

The authors stated that percent mass loss was greater for salamanders on top shelf than 

bottom shelf (authors state presumably due to temperature gradient), therefore there is 

uncertainty in the potential overall influence of different environmental conditions on the 

study results (the authors stated that they controlled for initial mass. Detailed 
174

descriptions on how the water conserving behaviors were observed is not reported (e.g., 

observed for organisms pressed against side of dish, was there a criteria for how much it 

must be touching dish; how long were the observations for each dish?). The results for 

percent against dish side was not dose responsive for two of the three trials suggesting 

variability in this behavior. For figure 2A it appear that the results for the 0 μg/L group 

(as triplets) is missing or the treatment groups overlay it to the extent it is masked. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
175

3.44. Rohr, J.R., T. Sager, T.M. Sesterhenn, and B.D. Palmer. 2006 

Chemical Name: Atrazine (Streamside Salamander Study; effects of atrazine exposure 

and post-exposure) 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 82038 

Rohr, J.R., T. Sager, T.M. Sesterhenn, and B.D. Palmer. 2006. Exposure, Postexposure, 

and Density-Mediated Effects of Atrazine on Amphibians: Breaking Down Net Effects 

Into Their Parts. Environ. Health Perspec. 114(1). p. 46-50. 

Purpose of Review: Registration Review

Date of Assessment: 5/1/12 (this review relies on review from 4/10/06 by Anita Pease, 

ERB3) 

Brief Summary of Study Findings: 

Streamside salamander embryos (Ambystoma barbouri) and larvae were exposed to 

atrazine (0, 4, 40, and 400 ppb) and survival was quantified during and 14 months after 

exposure. Atrazine-induced mortality during exposure was ameliorated by densitydependent survival after exposure, but complete density-dependent-mediated 

compensation was precluded by significant “carryover effects” of atrazine. Salamanders 

exposed to 4, 40 and 400 ppb of atrazine had significantly lower survival than did control 

animals 14 months (421 days) postexposure. The study author attempts to predict 

population-level effects as the sum of exposure effects, carryover effects (ie., effects of a 

pollutant may persist or appear after exposure), and density-mediated compensation (ie. 

If juvenile mortality occurs, survivors may experience less mortality because of reduced 

competition for resources). 

Methods: Eggs and larvae obtained from 2004 study by Rohr et al. (Rohr JR, Elskus AA, 

Shepherd BS, Crowley PH, McCarthy TM, Niedzwiecki JH, Sager T, Sih A, Palmer BD. 2004. Multiple 

stressors and salamanders: Effects of an herbicide, food limitation, and hydroperiod. Ecol Appl 14:1028–

1040. E 81748) After metamorphosis, A. barbouri from each aquarium were placed into 

terraria (18.5 cm in diameter, 7.5 cm high) so that the 48 aquarium replicates were 

preserved. Each terraria contained approx 6 cm of homogeneously mixed organic top 

soil. Salamanders were fed crickets. 

Description of Use in Document: Qualitative. 

Rationale for Use: Provides information about long-term survival of salamanders and 

provides information on terrestrial-phase amphibians. However, there were uncertainties 

identified with Rohr et al., 2004 which was where the organisms were exposed to 

atrazine. 
176

Limitations of the Study: The uncertainties in Rohr et al. 2004 include: uncertainties 

associated with the effects on embryos and recently hatched larvae because of the 

presence of a white film covering the embryo, suggesting a fungal pathogen, which may 

have caused decreased survival and increased time to hatching; effects on larval survival 

were different for different years (in 2002, there was no significant effect of atrazine on 

larval survival, whereas in 2003, larvae exposed to 400 ppb had significantly lower 

survival than control larvae). Metamorphic parameters for 2003 included outliers and 

were not included in the analyses. Duration of study not specified (~117 days). DMSO is 

not an acceptable solvent because it accelerates the movement of a chemical across cell 

membranes. In addition, no negative control (water only w/out DMSO) was included. 

Field collected organisms, therefore prior exposure is unknown; however, in another 

study by Rohr et al., 2003, at reported same collection site, they state that exposure from 

atrazine was likely given agricultural practices in area and in Rohr et al. 2004, the study 

reports an atrazine concentration of 33.9 ppb in a stream of same size as collection site in 

same state. 

Reviewer: Amy Blankinship, ERB3 

 
177

3.45. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. 

McCarthy, J.H. Niedzwiecki, T. Sagar, A. Sih, and B.D. Palmer. 2004 

Chemical Name: Atrazine (Streamside Salamander Study; effects of atrazine, food 

limitation, and hydroperiod) 

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 81748 

Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. 

Niedzwiecki, T. Sagar, A. Sih, and B.D. Palmer. 2004. Multiple Stressors and 

Salamanders: Effects of an Herbicide, Food Limitation, and Hydroperiod. Ecological 

Applications. 14(4). p. 1028-1040. 

Purpose of Review: Litigation and Registration Review

Date of Assessment: 4/10/06 updated 5/1/12 

Brief Summary of Study Findings: 

The study evaluated the combined effects of atrazine (80% ai) (approximate measured 

doses = 0, 4, 40, and 400 ppb), food limitation, and drying conditions on the survival, life 

history, and behavior of the streamside salamander (Ambystoma barbouri) from the 

embryo stage through metamorphosis. In general, food and atrazine levels did not 

interact statistically. Exposure to 400 ppb atrazine decreased embryo survival to Day 16 

and increased time to hatching. However, most embryo mortality was associated with a 

white film covering the embryo, suggesting the presence of a fungal pathogen. It is 

unknown whether the fungi caused or simply followed mortality. Delayed hatching 

could prolong time in streams and result in mortality from stream drying or from aquatic 

predation. Drying conditions and food limitation decreased larval survival, while 400 

ppb atrazine only reduced larval survival in one of the two years tested. The study author 

attributes the difference between the years in atrazine-related mortality to possible 

condition-dependent mortality. Sublethal effects included elevated activity and reduced 

shelter use associated with increasing atrazine conc and food limitation. Although 

atrazine-induced reduction in refuge use and increase in activity did not appear to 

strongly influence feeding rates, they may elevate predation risk by increasing 

conspicuosness and encounters with predators. Larval period was lengthened by food 

limitation and shortened by 400 ppb atrazine. Earlier metamorphosis may provide a 

benefit to atrazine-exposed animals by reducing exposure, but their smaller size at 

metamorph could result in lower terrestrial survival, lower reproduction and 

compromised immune function. Drying conditions accelerated metamorphosis for larvae 

exposed to 0 and 4 ppb atrazine, but did not affect metamorphosis timing for the 40 or 

400 ppb groups. Therefore, combined effects of stream drying and atrazine exposure 

may not pose a greater threat to salamander larvae than either factor alone. Food 

limitation, drying conditions, and 400 ppb of atrazine reduced size at metamorphosis 

without affecting body condition (relationship between mass and length), even though 
178

feeding rates did not differ significantly among atrazine concs at any time during 

development. This suggests that atrazine levels may have increased larval energy 

expenditures, reduced feeding duration due to a shortened larval period. Also may be 

related to neuroendocrine processes not related to activity and feeding. Larson study 

showed that atrazine at 250 ppb altered thyroxine levels of tiger salamanders, which 

reduced size at metamorphosis. 

The study author states that effects on metamorphosis were affected at 40 ppb. 

Methods: 31 eggs in 2002 and 40 eggs in 2003 placed into 48 aquaria (37 L); 12-h light: 

dark photoperiod, pH = 8, temp = 15 oC, 9.5 L water, with refuge plate in tank. Egg 

clutches collected from Fossil Creek (Jessamine County, KY). Four atrazine 

concentrations (all included solvent DMSO) at 0, 4, 40, and 400 ppb were tested and test 

solutions were renewed weekly. A factorial design (4 x 2 with 6 reps of each treatment 

combo) were used to cross 4 atrazine conc w/ 2 feeding regimes. Larvae in 1⁄2 aquaria fed 

live blackworms ad libitum (high food), other 1⁄2 were rationed 2.24 g 2x/wk (low food). 

Atrazine and food treatment was crossed with two hydroperiods (constant or lowered 

water level) in 4 x 2 x 2 factorial design (3 reps/treatment) with reduction in water level 

beginning at day 45. Water level was reduced by removing 1 L of water every other day 

until aquaria reached 2.5 L of water. 

Description of Use in Document: Qualitatively 

Rationale for Use: The study provides information on amphibian development and 

behaviors under different environmental conditions; however, there are uncertainties as 

described below. 

Limitations of the Study: There is uncertainty associated with the effects on embryos 

and recently hatched larvae because of the presence of a white film covering the embryo, 

suggesting a fungal pathogen, which may have caused decreased survival and increased 

time to hatching. Although pH, temp, and DO were adequate, nitrates and ammonia not 

reported and given the higher loading and with weekly renewal, there is uncertainty in 

whether ammonia levels were within acceptable limits 

Effects on larval survival were different for different years. In 2002, there was no 

significant effect of atrazine on larval survival, whereas in 2003, larvae exposed to 400 

ppb had significantly lower survival than control larvae. Metamorphic parameters for 

2003 included outliers and were not included in the analyses. 

Duration of study not specified (~117 days). No raw data provided. DMSO is not an 

acceptable solvent because it accelerates the movement of a chemical across cell 

membranes. In addition, no negative control (water only w/out DMSO) was included. 

Organisms were field collected, therefore prior exposure history is unknown. The study 

authors state that they do not have data on atrazine concentrations at collection site they 

state that an atrazine concentration of 33.9 ppb was detected in KY stream similar in size 
179

to stream where organisms were collected Additionally, these organisms were reported 

to be collected from same area as was reported in Rohr et al. 2003 (Environ. Toxicol. 

Chemistry 22 (10): 2385-2392). In that study, while they report they do not have 

information regarding atrazine concentrations from the collection site, they state that the 

organisms were likely exposed due to the agricultural practices surrounding the collection 

site. 

Reviewer: Anita Pease, ERB3; Amy Blankinship, Chemist, ERB3, Environmental Fate 

and Effects Division 

 
180

3.46. Rohr, J. R. and P. W. Crumrine. 2005 

Open Literature Review Summary 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Record Number and Citation: 

Rohr, J. R. and P. W. Crumrine. 2005. Effects of an herbicide and an insecticide on pond 

community structure and processes. Ecological Applications. 15(4): 1135-1147. (MRID 

48261130) 

Purpose of Review: 

Determine potential effects of atrazine on an aquatic community. Endpoints from this 

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant 

communities and associated determination of a Level of Concern (LOC). 

Date of Review: 

October 5, 2009 

Purpose of Study: 

This study examines the effect of the herbicide atrazine and the insecticide endosulfan 

(tested independently) on constructed outdoor freshwater pond communities when the 

presence of common community members (wood frog tadpoles, adult snails, and caged 

dragonfly larvae) was varied. 

Summary of Study Findings: 

The focus of the summary is on treatments with exposure to atrazine. 

Methods: 

Twenty wood frog (Rana sylvatica) clutches were collected in late February 2003 from 

Gray’s Arch Pond in Kentucky. Tadpoles were hatched and reared in outdoor wading 

pools (containing aged tap water and covered with 60% shade cloth). They were fed 

rabbit chow ad libitum. Clutches were thoroughly mixed prior to experimentation. Adult 

snails (Planorbella trivolvis) were collected in early March 2003 from a pond at the 

University of Kentucky’s Ecological Research Facility. They were raised in pond water 

in the laboratory until their use in the experiment. Late-instar dragonfly larvae (Anax 

junius) were collected in early March 2003 from various ponds at the University of 
181

Kentucky’s Ecological Research Facility. Prior to the experiment, the dragonflies were 

held individually in the laboratory and fed aquatic invertebrates and R. sylvatica tadpoles. 

The study authors employed a 3 × 2 × 2 × 2 completely randomized factorial design with 

three “pesticide” treatments (acetone solvent control, 25 μg/L of atrazine, and 10 μg/L of 

endosulfan), two tadpole treatments (presence or absence of 10 R. sylvatica tadpoles, 

Gosner stage 23-26), two snail treatments (presence or absence of 10 P. trivolvis), and 

two dragonfly treatments (presence or absence of one caged, late-instar A. junius). The 

experimental densities of the community members were at the high-end of regional 

densities (based on the personal observation of the study authors). The experiment lasted 

for four weeks; the entire design was replicated four times in four spatial blocks (96 

experimental units in total). No water (negative) control was included. According to the 

study authors, acetone was not expected to be toxic at the level used in the experiment 

(0.0246 ml acetone/L final concentration in the experimental tub was estimated by the 

secondary reviewer). 

Each experimental unit (mesocosm) was a polyethylene tub (11.3 L capacity, 40.6 × 28.5 

× 17.5 cm) containing a perforated, transparent plastic cup (0.296 L capacity) secured to 

one end of the tub with monofilament line and five microscope slides attached to the side 

of the tub with silicone. The tubs were located on gravel at the University of Kentucky’s 

Ecological Research Facility. One March 31, 2003, each tub received 9.0 L of aged tap 

water, 1.0 L of water from the pond from which the snails were collected, 15 g of leaves 

from the mixed deciduous forest surrounding the pond from which the wood frogs were 

collected, and 3 g of rabbit chow (to stimulate growth of periphyton). The pond water 

was the source of phytoplankton, zooplankton, and insect larvae/eggs. The tubs were 

covered with 60% shade cloth to prevent predation and allowed to mature for two weeks 

in order to establish a periphyton community prior to addition of the pesticide, tadpoles, 

snails, and dragonflies. 

On April 14, 2003, the three “pesticide” treatments (acetone control, atrazine, and 

endosulfan) were added to the tubs. For the solvent control, 246 μL of acetone was 

added to the appropriate tubs. For the atrazine treatment (25 μg/L), 27.8 μL of atrazine 

stock solution (8.984 mg atrazine per mL of acetone) was added. The atrazine was 99% 

pure. The concentration of the atrazine stock solution was verified by flame ionization 

detection gas chromatography. The tubs were redosed with atrazine two weeks later, 

midway in the experiment, in order to simulate a second run-off event. The maximum 

nominal concentration of atrazine was 50 μg/L (i.e., for the second two week period of 

the experiment). The authors do not state if a second dose of acetone was added to the 

acetone control tubs. Atrazine concentration was not monitored during the experiment. 

However, the study authors estimated that the nominal concentration was higher than the 

actual concentration due to heavy rainfall that occurred during the experiment. 
182

Tadpole, snail, and dragonfly masses were obtained before they were added into each tub 

(0.219 ± 0.002, 0.692 ± 0.007, and 0.960 ± 0.032 g/individual, respectively). Dragonflies 

were placed in the transparent, perforated cup of each tub so that they were nonlethal to 

tadpoles outside the cup but could be visually and chemically detected. Dragonflies were 

fed two randomly chosen R. sylvatica tadpoles from the wading pool stocks three times 

per week throughout the experiment. Empty cups were picked up and gently dropped to 

control for feeding disturbance across tanks. Dragonfly mortality was assessed during 

these feedings. 

Periphytic algae in each mesocosm were assessed by measuring the chlorophyll a

concentration of the periphyton scraped off a microscope slide. Chlorophyll a was 

measured using American Public Health Association standard protocols. Samples for 

chlorophyll measurements were taken just prior to treatment (all tubs) and then from half 

of the spatial blocks every other week throughout the experiment. Dissolved oxygen 

(DO) and pH measurements were taken weekly from each tub using meters (ICM Model 

31200 and Corning Model 320). 

Twice a week and once during the last week, the study authors conducted four 

observations on each mesocosm, recording the number of tadpoles observed (those not 

hiding under leaves), the proportion of tadpoles avoiding the cup (on the opposite half of 

the mesocosm), and the number of tadpoles moving during a 15-s interval. Metamorphs 

(Gosner stage 42-46) were observed and removed only during the last week of the 

experiment. The experiment was ended on May 12th for blocks 1 and 2 and on May 13th

for blocks 3 and 4. At experiment termination, surviving snails and tadpoles were 

counted and weighed and snail egg masses were counted for each mesocosm. Surviving 

tadpoles were euthanized with a 0.01% solution of benzocaine and preserved in 10% 

neutral buffered formalin for later determination of Gosner developmental stage. Water 

clarity was quantified by the study authors using a scale of 1 to 5. Chironomid larvae 

(dipterans) of the genus Polypedilum (periphytivores and detritivores) and cladoceran 

zooplankton of the genus Daphnia (primarily Daphnia pulex; phytoplanktivores) were 

the most common invertebrates in the mesocosms. Their abundance was quantified on a 

scale of 1 to 5. The study authors proposed that their introduction into the mesocosms 

was via the pond water aliquots. The presence of insects and crustaceans in genera other 

than Polypedilum and Daphnia were noted as the presence or absence of “other insects 

and crustaceans” (one lumped category for statistical analysis); these observations were 

rare. 

All statistical analysis was conducted using Statistica 5.5a, using the mesocosm as the 

experimental unit. Proportion data were angularly transformed. Covariates and block 

effects were included when significant. Three separate multivariate analyses of variance 

(MANOVA) were conducted to analyze tadpole behavior (% hiding, % observed 

avoiding cup, % observed moving), tadpole growth and development (mass/tadpole and 

Gosner stage), and snail growth and reproduction (mass per snail and egg masses per 

snail). Univariate analyses of variance (ANOVA) were subsequently performed on those 
183

parameters. DO and pH were regressed versus tadpole growth and development (mass 

per tadpole and Gosner stage) and snail growth and reproduction (mass per snail and egg 

masses per snail). One-way ANOVA was also used to assess the influence of the 

pesticide treatments on the duration of dragonfly survival. Repeated-measures ANOVA 

was used to evaluate the effect of pesticide treatments on chlorophyll a in the first and 

second half of the experiment. Treatment effects on the number of snails that died and 

the number of tadpoles that reached metamorphosis were evaluated using Poisson 

regression (PR; with a log link). Water clarity and the abundance of Polypedilum and 

Daphnia were analyzed using logistic regression for an ordinal multinomial distribution 

(OMR; with a logit link). The presence of other insects and crustaceans and dragonfly 

survival were evaluated using logistic regression for a binomial distribution (BR; with a 

logit link). When the main effect of the pesticide was significant (P < 0.05) in ANOVAs, 

the study authors performed a Turkey’s honestly significant difference multiple 

comparison test (Tukey’s hsd) for all pairwise comparisons. When the main effect of the 

pesticide was significant (P < 0.05) using logistic regression (PR, OMR, BR), the study 

authors performed Bonferroni-adjusted linear contrasts for all pairwise comparisons. 

Results: 

 Effects on Periphytic Algae 

Periphytic algae amounts were significantly reduced in mesocosms with atrazine 

treatment (5.575 ± 0.292 mg/m2

, mean ± SE) relative to the solvent control (6.575 ± 

0.411 mg/m2, mean ± SE, Tukey’s hsd, P < 0.001). 

 Effects on Water Clarity

According to the study authors, water clarity was greater in mesocosms with atrazine 

(OMR, P = 0.045), although it is unclear whether the statistical comparison was made 

relative to the solvent control or endosulfan treated mesocosms. 

 Effects on Insects and Crustaceans 

Relative to mesocosms containing solvent control, mesocosms with atrazine treatment 

had lower densities of Polypedilum larvae (OMR, P = 0.004). Atrazine treatment did not 

have a significant effect relative to the control on Daphnia abundance or % of 

mesocosms containing ‘other’ insects and crustaceans (Figure 1). 

Pesticide treatments (atrazine and endosulfan) did not significantly reduce the percentage 

of dragonfly larvae which survived until the end of the experiment (atrazine, 12.5%; 

endosulfan, 25%; solvent, 25%; BR, df = 2, 45, χ

2

 = 1.08, P = 0.582) or the mean number 

of days that dragonfly larvae were alive (atrazine, 17.50 ± 2.43 days; endosulfan, 21.56 ± 

1.96 days; solvent 22.13 ± 1.72 days; ANOVA, F2,45 = 1.60, P = 0.213). The study 

authors did not provide a statistical pairwise comparison between the atrazine treatment 

and the control for either dragonfly survival or mean number of days alive. 
184

Figure 1. Effects of pesticide treatments (atrazine, endosulfan, solvent control) on (A) Polypedilum

(chironomid) larvae abundance, (B) Daphnia abundance, and (C) the percentage of mesocosms 

containing insects or crustaceans other than Polypedilum and Daphnia. Bars represent means (± SE) 

of 32 mesocosms. Different lowercase letters above bars reflect significant differences at P < 0.05 

among treatments according to Tukey’s honestly significant difference multiple comparison tests. 

Figure 2 from Rohr and Crumrine, 2005. 
185

 Effects on Snails

Snail final mass was reduced by atrazine relative to the solvent control (Tukey’s hsd, P < 

0.001, Figure 2). When controlling for final mass, fewer egg masses per surviving snail 

were deposited in mesocosms with atrazine compared to the control; however, the 

difference was not significant (Tukey’s hsd, P > 0.05, Figure 3). 

Figure 2. (A) Effects of pesticide (atrazine, endosulfan, solvent control) and dragonfly treatments 

(presence and absence) on snail mass and (B) effects of pesticide and snail treatments (presence and 

absence) on tadpole mass. Values represent means and SE of eight mesocosms, and probability 
186

values are for the interaction between displayed treatments when controlling for significant 

predictors [see Tables 1 and 3 of Rohr and Crumrine (2005) for predictors]. Figure 4 from Rohr and 

Crumrine, 2005. 

Figure 3. Effects of pesticide treatments (atrazine, endosulfan, solvent control) on (A) the number of 

egg masses deposited per surviving snail and (B) tadpole Gosner development stage. Bars represent 

means (± SE) of 16 mesocosms. Different lowercase letters above bars reflect significant differences 

at P < 0.05 among treatments according to Tukey’s honestly significant difference multiple 

comparison tests when controlling for significant predictors [see Tables 1 and 3 of Rohr and 

Crumrine (2005) for predictors]. Figure 6 from Rohr and Crumrine, 2005. 

 Effects on Wood Frogs 

The study authors determined that the major temporal changes observed in wood frog 

behavioral responses through time were primarily due to dragonfly mortality. Since there 

were no significant treatment differences in dragonfly mortality or timing of mortality, 
187

the study authors used the mean of the seven days of wood frog behavior observations for 

subsequent analyses. A greater percentage of tadpoles were hiding in the solvent controls 

than the atrazine treatment (Tukey’s hsd, P = 0.028). Atrazine treatment did not have a 

significant effect on the number of tadpoles avoiding the cup compared to the control 

(Tukey’s hsd, P > 0.05, Figure 4). Atrazine treatment did not appear to affect tadpole 

activity relative to the control (Figure 5). Wood frog development (Gosner stage) was 

slowed by atrazine compared to the control but the difference was not statistically 

significant (Tukey’s hsd, P = 0.067, Figure 3). Atrazine treatment did not have a 

significant effect relative to the control on tadpole final mass (Tukey’s hsd, P > 0.05, 

Figure 2). 

Figure 4. Effects of pesticide treatments (atrazine, endosulfan, solvent control) on (A) the percentage 

of tadpoles hiding (measured as the percentage of total individuals that were not observed) and (B) 

the percentage of individuals observed that were avoiding the cup. Bars represent means (± SE) of 16 

mesocosms. Different lowercase letters above bars reflect significant differences at P < 0.05 among 

treatments according to Tukey’s honestly significant difference multiple comparison tests when 
188

controlling for significant predictors [see Table 2 of Rohr and Crumrine (2005) for predictors]. 

Figure 7 from Rohr and Crumrine, 2005. 

Figure 5. Interaction among pesticide (atrazine, endosulfan, solvent control), snail, and dragonfly 

treatments (presence and absence) on the percentage of tadpoles observed that were moving during a 

15-s observation period. Values represent means (± SE) of four mesocosms. All main effects and twoway interactions were significant (P < 0.05) when controlling for Daphnia abundance [see Table 2 of 

Rohr and Crumrine (2005)]. Figure 9 from Rohr and Crumrine, 2005. 

Data Summary Table: 

Effect 

Magnitude 

of Effect 

relative to 

controls 

Nominal 

Atrazine 

Concentration 

(μg/L) 

Day of Study 

Effect 

Occurred 

Time Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Chlorophyll a 15% 

decrease 

50 μg/L 

(maximum)1

Mean of data 

collected over 

the four week 

study period 

Could not be 

determined. 

Could not 

be 

determined. 

Mean 

atrazine, 

(5.575 

mg/m2

); 

mean 

solvent, 

(6.575 

mg/m2

). 

Tukey’s hsd, 

P < 0.001 

Polypedilum

abundance 

score 

~24% 

decrease 

50 μg/L 

(maximum)1 Unknown2 Could not be 

determined. 

Could not 

be 

determined. 

Figure 1 

OMR, P = 

0.004 

Daphnia

abundance 

No effect 

observed 

50 μg/L 

(maximum)1 Unknown2 Could not be 

determined. 

Could not 

be 

Figure 1 
189

Effect 

Magnitude 

of Effect 

relative to 

controls 

Nominal 

Atrazine 

Concentration 

(μg/L) 

Day of Study 

Effect 

Occurred 

Time Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

determined. Tukey’s hsd, 

P > 0.05 

Other insects/ 

crustaceans 

abundance 

No effect 

observed 

50 μg/L 

(maximum)1 Unknown2 Could not be 

determined. 

Could not 

be 

determined. 

Figure 1 

Tukey’s hsd, 

P > 0.05 

Dragonfly 

larvae survival 

50% 

decrease 

50 μg/L 

(maximum)1

 Survival 

through the 

end of the 

experiment 

(days 29/30)3

Could not be 

determined. 

Could not 

be 

determined. 

Atrazine 

(12.5%); 

solvent, 

(25%) 

Unknown if 

difference 

was 

significant 

(no 

statistical 

comparison 

provided), 

variation 

among 

replicates 

unknown 

(only mean 

values 

provided). 

Dragonfly 

larvae lifespan 

21% 

decrease 

50 μg/L 

(maximum)1

Mean of data 

collected over 

the four week 

study period. 

Could not be 

determined. 

Could not 

be 

determined. 

Atrazine 

(mean ± SE; 

17.50 ± 

2.43); 

solvent 

(mean ± SE, 

22.13 ± 

1.72). 

Unknown if 

difference 

was 

significant 

(no 

statistical 

comparison 

provided), 

SE values 

suggest that 

means may 

not be 

significantly 

different. 
190

Effect 

Magnitude 

of Effect 

relative to 

controls 

Nominal 

Atrazine 

Concentration 

(μg/L) 

Day of Study 

Effect 

Occurred 

Time Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Snail final 

mass 

Reduction 50 μg/L 

(maximum)1

Days 29/30 

(study 

termination)3

Could not be 

determined. 

Could not 

be 

determined. 

Figure 2 

Tukey’s hsd, 

P < 0.001 

(text). 

No. egg mass 

per snail 

No effect 

observed 

50 μg/L 

(maximum)1

Days 29/30 

(study 

termination)3

Could not be 

determined. 

Could not 

be 

determined. 

Figure 3 

Tukey’s hsd, 

P > 0.05 

Tadpole final 

mass 

No effect 

observed 

50 μg/L 

(maximum)1

Days 29/30 

(study 

termination)3

Could not be 

determined. 

Could not 

be 

determined. 

Figure 2 

No effect 

judged by 

overlapping 

error bars 

for atrazine 

treatment 

and control. 

No 

statistical 

comparison 

provided 

Tadpole 

Gosner stage No effect 50 μg/L (maximum)1

Days 29/30 

(study 

termination)3

Could not be 

determined. 

Could not 

be 

determined. 

Figure 3 

Tukey’s hsd, 

P = 0.067 

(text) 

Tadpoles 

hiding 

~ 6% less 

hiding 

50 μg/L 

(maximum)1

Mean of data 

collected over 

the four week 

study period. 

Could not be 

determined. 

Could not 

be 

determined. 

Figure 4 

Tukey’s hsd, 

P = 0.028 

(text) 

Tadpoles 

avoiding cup No effect 50 μg/L (maximum)1

Mean of data 

collected over 

the four week 

study period. 

Could not be 

determined. 

Could not 

be 

determined. 

Figure 4 

P > 0.05 
191

Effect 

Magnitude 

of Effect 

relative to 

controls 

Nominal 

Atrazine 

Concentration 

(μg/L) 

Day of Study 

Effect 

Occurred 

Time Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Tadpoles 

moving/ 

mobility 

No effect 

observed 

50 μg/L 

(maximum)1

Mean of data 

collected over 

the four week 

study period. 

Could not be 

determined. 

Could not 

be 

determined. 

Figure 5 

No effect 

judged by 

overlapping 

error bars 

for atrazine 

treatment 

and control. 

No 

statistical 

comparison 

provided 1

 Initial dose of atrazine was 25 μg/L, (nominal) then after two weeks the ponds were redosed with 25 μg/L 

(nominal). Thus the exposure for the first two weeks was 25 μg/L (nominal) and the exposure for the 

second two weeks was assumed by the study authors to have been a maximum of 50 μg/L (nominal). For 

the purposes of the associated endpoint summary spreadsheet, it was assumed that 25 μg/L was the initial 

test concentration. 

2

 Not specified by study authors. Primary reviewer assumed study termination (day 29/30). 

3

 Experiment was terminated in half of the ponds on day 29 and the other half on day 30. 

Reviewer Comments: 

Much of the study results as presented by the study authors were not specific to atrazine 

treatment effects. Results of MANCOVA, ANCOVA, ANOVA, and Poisson regression 

did not distinguish the individual effects of atrazine. In general, these analyses examined 

the effects of “pesticide” treatment (atrazine, endosulfan, or solvent control) or the effects 

of non-pesticide treatments (e.g. effects of snail absence or presence on chlorophyll a

with the “pesticide” treatments combined). The study authors presented the simple effect 

of atrazine treatments for some but not all results, that is, a pairwise comparison between 

atrazine treated and solvent control mesocosms. This study review focuses on and 

presents results specific to atrazine treatment effects, unless otherwise noted (i.e., 

analyses and results that include endosulfan treatments are generally excluded). It should 

also be noted that mesocosms treated with atrazine (endosulfan and control as well) 

varied in regards to the presence or absence of snails, dragonflies, and tadpoles, and that 

these non-pesticide treatment effects were integrated into the atrazine treatment effects 

presented in this study. 

The study authors stated that direct effects of atrazine (i.e., reduction of periphyton 

chlorophyll a) resulted in indirect effects on snails, tadpoles, and Polypedilum

chironomid larvae. 
192

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the weight of evidence regarding effects of 

atrazine on aquatic plant communities.

Limitations of Study: The results have good applicability to mesocosms or natural 

aquatic systems since the experimental mesocosms contained not only periphytic algae, 

but also several species from higher levels of the food web, however, there were 

limitations. Actual pesticide (atrazine) concentrations were not monitored throughout the 

experiment. Although a nominal dose was reported, exposure was complicated because a 

second dose of 25 μg atrazine/L was made two weeks after the initial application of 25 

μg/L and the study authors reported heavy rainfall (timing not reported) during the 

experiment which would have diluted the pesticide in the mesocosms. Nominal 

concentrations were verified in the stock solution and atrazine should have been stable 

over a period of two weeks; therefore, the study authors made a conservative assumption 

that 50 μg atrazine/L was in each mesocosm It was not clear if the solvent control pond 

received a second application of acetone after two weeks; effects of acetone would have 

been underestimated if a second application was not made. There was no effort to 

determine if atrazine or other contaminants were present in the source water for the 

experiments or the control mesocosms. Although there was an acclimation period for the 

periphyton, it does not appear that there was an acclimation period for the snails, 

tadpoles, and dragonflies. In addition, it is unclear whether wood frog tadpoles, adult 

snails, and dragonfly larvae may have been previously exposed to other contaminants 

(including pesticides) prior to the experiment, given that no previous contaminant history 

of the collection sites was provided. 

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc 

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA 

 
193

3.47. Rohr, J. R., A. M. Schotthoefer, T. R. Raffel, H. J. Carrick, N. 

Halstead, J. T. Hoverman, C. M. Johnson, L. B. Johnson, C. Lieske, M. D. 

Piwoni, P. K. Schoff, and V. R. Beasley. 2008. 

Open Literature Review Summary 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Record Number and Citation: 

Rohr, J. R., A. M. Schotthoefer, T. R. Raffel, H. J. Carrick, N. Halstead, J. T. Hoverman, 

C. M. Johnson, L. B. Johnson, C. Lieske, M. D. Piwoni, P. K. Schoff, and V. R. Beasley. 

2008. Agrochemicals increase trematode infections in a declining amphibian species. 

Nature. 455(7217): 1235-1239. (MRID 48261131) 

Purpose of Review: 

Determine potential effects of atrazine on an aquatic community. Endpoints from this 

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant 

communities and associated determination of a Level of Concern (LOC). 

Date of Review: 

October 10, 2009 

Purpose of Study: 

This study examines the relationship between environmental concentrations of the 

herbicide atrazine and the number of trematode infections in the declining northern 

leopard frog Rana pipiens by studying the effects of atrazine on R. palustris and R. 

pipiens in prepared community-level mesocosms and by conducting a wetland survey to 

identify the best predictors of total larval trematode abundance in R. pipiens. 

Summary of Study Findings: 

The focus of the summary is on the mesocosm experiment. Methods and results from the 

wetland survey are briefly mentioned. 

Methods: 

Mesocosm experiment 
194

The experiment was conducted at Pennsylvania State University using a randomized 

block design. The Pennsylvania State University area was in the southern range of R. 

pipiens where, according to the study authors, the species is declining. The test container 

was a 1,100-L plastic cattle tank filled with 800 L of water and covered with shade cloth. 

Twelve tanks were prepared for the test (atrazine = 4, controls = 8). Three weeks prior to 

dosing, each tank was prepared with 300 g of mixed hardwood leaves, plexiglass to 

sample snail egg masses and hatchlings, periphyton samplers, and inoculations of 

zooplankton, periphyton, and phytoplankton from four ponds (location of ponds not 

reported). Just before dosing, each test tank was prepared with four larval amphibians (5 

Ambystoma maculatum, 20 Hyla versicolour, 20 Rana palustris and 20 Rana clamitans), 

two snail (11 Planorbella trivolvis and 10 Physa gyrina), one beetle (5 Hydrochara sp.), 

two water bug (2 Belostoma flumineum and 7 Notonecta undulate) and one dragonfly 

species (2 Anax junius). The R. palustris (pickerel frog) and R. clamitans (green frog) are 

congeners of the declining northern leopard frog R. pipiens. All snails which were used 

in the experiment were screened twice for patent (shedding) trematode infections before 

being placed in the tanks. Tanks were exposed to atrazine via a single dose of technical 

grade atrazine (from ChemService; % purity was not reported) at 102 μg/L. According to 

the study authors, 102 μg/L atrazine was chosen as the expected environmental 

concentration, based on calculations using the U.S. EPA’s GENEEC (version 2) 

software. One hour after dosing, water samples were taken and shipped on ice for 

analysis of actual applied atrazine (mean 117 μg/L). In order to simulate exposure to 

trematodes, each tank also received a caged planorbid snail shedding cercariae from the 

family Plagiorchidae (the timing of this addition was not reported). The caged snails 

were rotated among the tanks every other day for homogenization. Snails were 

thoroughly rinsed between tanks; tanks were not rotated. The experiment ran for 4 

weeks (one generation) from the end of June 2007 to July 2007. Both water and solvent 

control tanks were prepared (unreported number of tanks per control). The solvent used 

and its final concentration were not reported. 

 

At the end of the experiment, the study authors took water samples for phytoplankton 

analyses, scraped periphyton from the periphyton samplers, and scored water clarity on a 

scale of 1 to 5. Additionally, the study authors counted the snail egg masses on the walls 

of each tank, the snails hatchlings from the plexiglass placed in each tank and the 

surviving and dead animals. Chlorophyll a measurements were made using standard 

fluorometry methods (not specified). Five preserved R. clamitans and five R. palustris

tadpoles from each tank were selected randomly and melanomacrophage and eosinophils 

cells were counted per field of view from their sectioned and stained livers (zero-to-five 

scale, where zero equated to none and five equated to more than 90% of the liver 

contained melanomacrophage or eosinophils cells). Each tadpole was also cleared and 

stained and their larval trematodes were counted under a compound scope (zero-to-five 

scale). All variables were quantified blind to the mesocosm treatment. The mean of the 

mesocosm unit data was used for statistical analysis. 

The effect of atrazine on chlorophyll a concentration in phytoplankton and periphyton 

and on percent survival (after angular transformation) was analyzed using the general 
195

linear model. Log eosinophils and log melanomacrophages per field of view and 

trematode loads were analyzed using the general linear model controlling for the biomass 

of plagiorchid-infected snails per tank. In leukocyte and trematode load analyses for R. 

clamitans only, the study authors controlled for mean Gosner development stage due to 

the fact that their previous work demonstrated that susceptibility to trematodes changes 

can occur in different developmental stages for this species. The effect of atrazine on 

counts of snail egg masses and snail hatchlings was analyzed using the generalized linear 

model with a Poisson error distribution and a log link. The effect of atrazine on water 

clarity was analyzed using the generalized linear model with an ordinal multinomial error 

distribution and a logit link. The physid snail data was not analyzed due to lack of 

experimental data. For each of the mesocosm analyses, the tank was the replicate and the 

block was included in the model if its probability value was less than 0.25. To determine 

if eosinophils and melanomacrophages were significant predictors of plagiorchid loads, 

the study authors conducted a regression analysis for each predictor using individual 

tadpoles as replicates and blocking by tank. The water and solvent control tanks were 

pooled for analyses because no significant difference was found between these 

treatments; however, the study authors did not specify the statistics used and if the lack of 

significance was true for all response variables. 

The study authors rescreened the snails at the end of the experiments and noted that some 

were pre-patent (infected but not shedding trematodes) at the beginning of the experiment 

and began shedding plagiorchid cercariae during the experiment. Upon dissection, the 

study authors discovered that one of the snails was infected with strigeid trematodes; 

therefore, the tank from which the snail was taken was eliminated from the trematode 

load analyses. 

Wetland survey

Eighteen (18) wetlands in the Broadleaf Forest Ecoregion in Minnesota, USA, were 

surveyed regarding more than 240 plausible predictors of the abundance of larval 

trematodes in northern leopard frogs (R. pipiens) living in those wetlands. During 1999, 

the study authors characterized biological diversity (three visits: March-April, May-June 

and, July-August) and obtained water, sediment, and amphibian tissue samples to 

quantify various analytes (two visits: April-May and June-July). Analyte quantification 

was conducted using standard US EPA protocols. During each visit to the wetland, the 

study authors attempted to collect 15 recently metamorphosed R. pipiens for parasite 

quantification and 25 for pathology. Faunal diversity was conducted using daytime, 

visual time and area-constrained dip-net sampling methods with effort adjusted according 

to wetland size. Relative plant cover of each plant species was estimated using three 10-

m transects and the line-intercept method. Landscape structure was summarized at 1-km 

and 10-km extents using Fragstats. Frogs were inspected for parasites (buccal and 

abdominal cavities, visceral organs, skin, and eyes). After necropsy, a dissecting 

microscope was used to identify encysted metacercariae. Frog livers (frogs in Gosner 

stage 46) were examined for melanomacrophage and eosinophils cells under a light 

microscope. 
196

The statistical analysis for the wetland survey was done in four steps: 1) calculated the 

coefficient of determination (R2

) of each predictor in the absence of covariates; 2) used 

the general linear model to generate the 20 best subset models for mean larval trematode 

loads in R. pipiens (maximum of 3 predictors, ranked by R2

); 3) used a structural 

equation model, followed by path analysis to evaluate the amount of support for the 

specific proposed casual mechanisms underlying the significant positive relationship 

between atrazine concentration and larval trematode abundance; and 4) assessed whether 

atrazine was correlated with any other variables that might explain the results more 

concisely. Atrazine and desethylatrazine (one of the transformation products of atrazine) 

were measured separately; however, they were combined together as “atrazine” for the 

analysis since their combination was the best predictor of total larval trematode 

abundance in R. pipiens. 

Results: 

 Mesocosm Experiment 

In the mesocosm experiment, atrazine significantly reduced phytoplankton chlorophyll a

concentrations (mean ± s.e.m.; atrazine treatment, 2.52 ± 0.45; water and solvent 

controls, 7.14 ± 1.67; general linear model, F = 4.88, P = 0.031). The study author’s 

state that this reduction resulted in greatly increased nutrient availability, water clarity, 

and sunlight penetration to attached algae, however, only data on water clarity are 

reported (Table 1). The concentration of periphyton chlorophyll a was significantly 

elevated in the atrazine-treated mesocosms (1.04 ± 0.18), compared to the water and 

solvent controls (0.70 ± 0.08; general linear model, F = 7.17, P = 0.032). 

The snail population of the atrazine-treated tanks was more than four times that of the 

control tanks in just one generation (P < 0.001). 

For R. palustris, specimens from the atrazine-treated mesocosms showed much lower 

liver melanomacrophages and eosinophil counts than specimens from the control tanks. 

Survival of R. palustris was also much lower in the atrazine-treated tanks (47.5% survival 

in the atrazine treatment group versus 70% survival in the controls), preventing the study 

authors from performing an unconfounded test of the effect of atrazine on trematode 

loads (since the most infected individuals were most likely those which did not survive). 

For R. clamitans, the study authors did not observe significant differences between the 

atrazine-treated tanks and the control tanks regarding liver melanomacrophages or 

elevated mortality; however, atrazine-treated mesocosms showed significant reductions 

in liver eosinophil counts. R. clamitans in atrazine treated tanks had significantly more 

plagiorchid trematodes than in control tanks when controlling for the relationship 

between plagiorchid loads and eosinophils. Plagiorchid trematode infections were not 

reported for R. palustris. 
197

The findings of the mesocosm experiment and statistic analysis results are reported in 

Table 1. 

Table 1. Findings from mesocosm experiment examining the effect of atrazine (mean ± s.e.m.) versus 

the control (water and solvent combined, mean ± s.e.m.) on factors proposed to influence larval 

trematode loads in frogs. Table 1 from Rohr et al. 2008.

Response variable Atrazine1 Control2 Test statistic P3

Chlorophyll a in 

phytoplankton (μg/L) 2.52 ± 0.45 7.14 ± 1.67 4.884

 0.0315

Chlorophyll a in periphyton 

(mg/m2

) 1.04 ± 0.18 0.70 ± 0.08 7.174

 0.032 

Water clarity (scale 1-5) 4.75 ± 0.25 3.88 ± 0.23 5.256 0.0115

Snail hatchings (per m2) 468.09 ± 205.85 110.64 ± 29.31 20.197 <0.001 

Snail egg masses (per m2) 30.45 ± 3.03 12.37 ± 4.34 4.987 0.026 

Melanomacrophages per field 

of view for R. palustris (log) 0.16 ± 0.06 0.36 ± 0.06 4.504

 0.0315

Eosinophils per field of view 

for R. palustris (log) 0.03 ± 0.14 0.21 ± 0.06 1.884

 0.1025,8

Survival of R. palustris (%) 47.50 ± 7.50 70.00 ± 3.90 9.774 0.017 

Melanomacrophages per field 

of view for R. clamitans (log) 0.25 ± 0.13 0.38 ± 0.09 0.674

 0.2175,9

Eosinophils per field of view 

for R. clamitans (log) 0.11 ± 0.10 0.39 ± 0.07 4.754

 0.0295

Survival of R. clamitans (%) 76.25 ± 3.15 76.25 ± 2.80 <0.0014 0.97110

No. of plagiorchid trematode 

cysts in R. clamitans 20.61 ± 4.52 7.51 ± 3.30 4.904

 0.0315,11

1

 n =4. 

2

 n=8; the solvent and water controls were pooled because there was no significant difference between the 

two treatments (details of statistical testing not reported). 

3

 Block was included in the model if its P value was less than 0.25. 4 F statistic from analyses using the general linear model. 5

 One-tailed test. 

6 χ2

 statistic from analyses using the generalized linear model with an ordinal multinomial error distribution 

and logit link. 

7 χ2

 statistic from analyses using the generalized linear model with a Poisson error distribution and a log 

link. 

8

 The power of the test was 0.646; the minimum sample size necessary per group to detect a significant 

difference given the power was six. 

9

 The power of the test was 0.326. 

10 The power of the test was 0.056. 

11 Controlling for biomass of plagiorchid-infected snails (F1,7 = 5.45, P = 0.026) and mean log eosinophil 

counts in tadpoles (F1,7 = 3.58, P = 0.050). 

 Wetland Survey
198

The study authors’ analysis of 18 wetlands in Minnesota, USA, indicated that atrazine 

combined with desethylatrazine (one of its transformation products) was the best 

predictor of total larval trematode abundance in R. pipiens out of more than 240 

measured plausible predictors of amphibian trematode infections. Atrazine alone (R2

 = 

0.46, F1,15 = 12.77, P = 0.003, see Figure 1) and desethylatrazine alone (R2

 = 0.46, F1,15 = 

12.93, P = 0.003) were significant predictors of increased trematode infections in R. 

pipiens. The sum of atrazine and desethylatrazine accounted for more variation than any 

other single predictor (51%, Monte Carlo randomization test, P = 0.001; see Figure 2a). 

Atrazine combined with desethylatrazine was a significant negative predictor of 

melanomacrophages aggregate scores (R2 = 0.17, F1,16 = 3.17, P = 0.047; See Figure 3). 

Melanomacrophage scores were from 0 (not present) to 5 (> 90% of liver filled with 

melanomacrophage aggregates). 

Figure 1. The relationship between larval trematode abundance in northern leopard frogs, Rana 

pipiens, and detectable concentrations of atrazine. Each plotted point represents a wetland and 95% 

confidence bands are shown. Figure S3A from Rohr et al., 2008. 
199

Figure 2. Proposed mechanisms for the relationship between atrazine and larval trematodes in 

amphibians. a, The relationship between detectable concentrations of atrazine plus desethylatrazine 

in water samples and larval trematode abundance (‘probit’ transformed, that is, the normal 

standard deviate of mean abundance/1,000) in northern leopard frogs, R. pipiens. Each data point 

represents a wetland, and a 95% confidence band is shown. b, Results of a path analysis examining 

the suggested mechanisms by which atrazine plus desethylatrazine increase larval trematode 

abundance. Unstandardized and standardized coefficients, respectively, are provided next to each 

path. Solid arrows represent significant paths. The model chi-square was 0.339 (d.f. = 1, P = 0.561), 

indicating that the model was a good fit to the data. Figure 1 from Rohr et al., 2008. 
200

Figure 3. The relationship between melanomacrophage aggregate score in R. pipiens and 

concentrations of atrazine plus desethylatrazine in water samples. In the graph and analyses, the 

study authors used the residuals from the relationship among mean melanomacrophage aggregate 

scores and the richness and abundance of nematodes and adult trematodes in leopard frogs. Each 

data point represents a wetland, and 95% confidence bands are shown. Individual data for atrazine 

was not illustrated. Figure 2a from Rohr et al., 2008.

 

The study authors also noted that larval trematode loads in amphibians must depend on 

the abundance of definitive hosts, such as amphibian-eating birds (a source of trematode 

eggs in wetlands). This dependence was supported by the significant interaction between 

atrazine and the number of suitable host habitat patches (forest, wetlands and open water) 

around each wetland (Monte Carlo randomization test: 1 km radius, P = 0.004; 10 km 

radius, P = 0.019). 

The analyses for the wetlands survey were mainly based on atrazine combined with 

desethylatrazine in the study report. However, in the supplemental information (found 

online at http://www.nature.com/nature/journal/v455/n7217/extref/nature07281-s1.pdf), 

some results were presented for atrazine and desethylatrazine individually. In addition, 

supplementary details about the models and results from the models are presented. 

 Overall Results 

Overall, based on the results of the study, the study authors concluded that atrazine (plus 

desethylatrazine) can be a primary driver of larval trematode infections for a declining 
201

amphibian species at concentrations commonly occurring in freshwater ecosystems (they 

also suggested that phosphate played a complementary role). The study authors proposed 

that atrazine increased exposure and susceptibility to larval trematodes by increasing the 

amounts of snail intermediate hosts (via increasing the periphyton) and by suppressing 

amphibian immune responses. The scheme of the proposed path of atrazine to increased 

larval trematode infections and mortality in amphibians is illustrated in Figure 4. 

 
202

Figure 4. A simple scheme which summarizes the main findings of the study authors. Figure S4 from 

Rohr et al., 2008. 

Data Summary Table: 
203

Effect 

Magnitude of 

Effect relative 

to controls 

Nominal 

Atrazine 

Concentration 

(μg/L)1

Day of 

Study Effect 

Occurred 

Time 

Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

When Effect 

Occurred 

Did 

Recovery 

Occur – if 

so, when? 

Comments 

Chlorophyll a in 

phytoplankton 65% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0312

Chlorophyll a in 

periphyton 49% higher 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0322

Snail hatchings 323% higher 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P < 0.0012

Snail egg 

masses 146% higher 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0262

Melanomacrophages per field 

of view for R. 

palustris 

56% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0312

Eosinophils per 

field of view for 

R. palustris 86% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.1022

Survival of R. 

palustris 32% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0172

Melanomacrophages per field 

of view for R. 

clamitans 

34% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.2172

Eosinophils per 

field of view for 

R. clamitans 72% lower 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0292

Survival of R. 

clamitans No effect 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.9712

No. of 

plagiorchid 

trematode cysts 

in R. clamitans

174% higher 102 μg/L 

Four weeks, 

study 

termination. 

Could not be 

determined. 

Could not be 

determined. 

P = 0.0312

1

 Mean actual concentration was 117 μg/L. Samples taken one hour after application; however, samples 

were sent to another lab to be analyzed at an unknown time after application. 

2

 See Table 1 for information about statistics. 

Reviewer Comments: (on mesocosm experiments) 
204

Several details of the mesocosm methodology were missing. Given that this study was a 

“Letters” communication in the journal Nature, it was expected that the study report and 

methodology would be abbreviated. However, no details were provided regarding the 

water used for the mesocosm experiment (the pond characteristics and locations of the 

four ponds were not specified; freshwater source or otherwise). The solvent used and its 

final concentration were not reported. In addition, it is unclear whether the larval 

amphibian, snail, beetle, water bug, and dragonfly species may have been previously 

exposed to other contaminants (including pesticides) prior to the experiment, given that 

no previous contaminant history or the location of the collection sites was provided. 

There was a three week acclimation period for zooplankton, phytoplankton, and 

periphyton; however, all other taxa were added to the tanks just before dosing with 

atrazine. 

Atrazine concentrations were not monitored throughout the experiment. Mesocosms 

were sampled one hour after dosing and shipped to another lab for measurement of 

atrazine. It was not stated how long after sampling that atrazine was actually measured. 

It was not reported if atrazine was present in the source ponds or the control mesocosms. 

It was not reported if the algal communities (i.e. chlorophyll a concentrations) were 

similar among the mesocosms prior to dosing. 

The generalized linear model was used to test for significant differences between the 

atrazine treatment and the controls. The study authors did not explain why this analysis 

was chosen instead of a simpler statistical method for comparing two treatments. 

 

The study authors suggested that the potentially important relationship between atrazine 

(pollution) and amphibian parasitism would not have been detected in the standard tests 

conducted in the United States and Europe for chemical registration since the tests are 

typically conducted on solitary species (isolated from other species with which they 

coexist, such as their parasites). 

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the weight of evidence regarding effects of 

atrazine on aquatic plant communities.
205

Limitations of Study: Although the experimental mesocosms contained both 

phytoplankton and periphytic algae, as well as several species from higher levels of the 

food web (i.e., larval amphibian, snail, beetle, water bug, and dragonfly species); there 

were a number of limitations associated with the study. The study authors did not report 

the solvent or the concentration in which it was used. Actual pesticide (atrazine) 

concentrations were not monitored throughout the 4-week experiment. Water samples 

collected one hour after dosing measurement of atrazine concentration; however, samples 

were shipped to another lab and it is unclear when the analysis occurred. The source 

ponds and the control mesocosms were not analyzed for atrazine, and the contaminant 

history of locations where the animals were collected, prior to the experiment, was not 

provided. In addition, it is unknown if the algal communities were similar among the 

mesocosms prior to dosing. 

Primary Reviewer: Lisa Muto, M.S., Staff Scientist, Cambridge Environmental Inc 

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA 

 
206

3.48. Rohr, J. R., A. Swan, T.R. Raffel, and P.J. Hudson. 2009. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Rohr, J. R., A. Swan, T.R. Raffel, and P.J. 

Hudson. 2009. Parasites, info-disruption, and the ecology of fear. Ocecologia. 159: 

447-454. (ECOTOX Ref. No. 11715) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/1/12 

Summary of Study Findings: 

The objective of the study was to evaluated whether atrazine was an info-disruptor in 

amphibians, altering their perception (responses to) of risk of predation and parasitism. 

Methods 

American toad, Bufo americanus tadpoles (Gosner stage 25-27; 6/cup containing 500mL 

dechlorinated water) were exposed to either 201 μg/L atrazine (99% ai, dissolved in 

acetone) (n=6 cups) or an acetone solvent control (0.0002%) (n=6 cups) for 4 days under 

static conditions. Tadpoles held at 20°C and fed fish flakes and frozen spinach for first 3 

days and no food on 4th and after 4 days survival was reported at nearly 100%. Tadpoles 

were collected shortly after hatching from a pond in Center County, Pennsylvania in 

which the authors reported was isolated from agricultural activity and atrazine inputs. 

The authors reported using a 2x6 completely randomized block design: tadpoles exposed 

to atrazine crossed by one of six cues: ASW (artificial spring water); an uninfected 

Planorbella trivolvis snail; a P. trivolvis snail releasing Echinostoma trivolvis cercariae; 

E. trivolvis cercariae alone; fish flskes (food resource); or two macerated conspecifics 

(predation cue). All 12 treatments were tested each day; ten experimental blocks; no 

tadpole was tested more than once. Behavioral cue experimental apparatuses (1m long 

gutters marked every 5 cm to create 20 segments) filled with 3.5L were randomly 

assigned to one of the 12 cups containing tadpoles. Three tadpoles removed from the cup 

(placed into a nitex cage) for each gutter/trial and tadpoles allowed to swim freely in 

gutter; authors state allowed for 10 replicates/treatment; one replicate/day; with each 

replicate containing 3 tadpoles. Cercariae were collected from two E. trivolvis infected 

snails; the same two snails were used as the infected snails during the trials. Cercariae 

maintained in plastic cylinder covered in Nitex mesh. Nitex mesh cages also used to 

contain snails, fish flake food and macerated conspecifics (macerated American toad 

tadpoles) and the cages were placed into one end of a gutter with an empty cage at the 
207

other end of the gutter. Black window screens placed in front of cages to prevent 

tadpoles from seeing cues or hiding under cages. Tadpoles had a 30 minute pre and post 

cue acclimation period and total of ten trials were conducted which were recorded with 

overhead digital camcorders. Tadpoles were exposed to cues for one hour and from the 

video, the location of each tadpole was recorded every 2 minutes which the author stated 

was to evaluate whether tadpole was attracted to, or avoided the cues. The number of 

lines the tadpole crossed was also measured; an average distance and number of lines 

were used in the statistical analysis. 

Results 

Measured concentration of atrazine stock was 196 μg/L. The study authors state that 

tadpoles were significantly closer to food cages (p=0.026) and significantly avoided [by 

distance from cage/cue] macerated conspecifics (p=0.004), a snail shedding cercariae 

(p=0.022) and cercariae alone (p=0.004) but did not avoid [based on distance] uninfected 

snail. There was no significant difference between responses for infected snail, cercariae 

alone, or macerated conspecifics. In addition, previous exposure to atrazine did not 

significantly affect responses for any of the cues. Cercariae alone and infected snail 

treatments significantly increased tadpole activity (by number of segments crossed) 

(p<0.044); but other treatments not significantly different including previous exposure to 

atrazine (Figure 1). 

Description of Use in Document (QUAL, QUAN, INV):
208

Qualitative 

Rationale for Use: 

The study provides information about amphibian tadpole behaviors to a variety of cues at 

one atrazine concentration. It also provides information on the survival of tadpoles at 

atrazine concentrations of 196 ppb. 

Limitations of Study: 

The study provides information on the survival of tadpoles to atrazine for 4 days, 

however, the results of the behavioral studies cannot be used for the risk assessment 

endpoints of survival, growth or reproduction as direct measures of these endpoints were 

not conducted (exposures were for one hour only). 

While the authors state that tadpoles collected from site isolated from agricultural 

activity, there may have been exposure to other chemicals present. Analysis of food 

source for potential contaminants not reported. Other than temperature, water quality 

parameters were not reported; test vessel material not reported. Although the study author 

states that previous work showed no effect from solvent, there is some uncertainty in the 

exclusion of a negative control. 

The same seven infected snails were used during the study in which the average number 

of cercariae were measured over the course of two days. On each of the 10 trial days, 

two [out of the seven] snails were used to collected cercariae and also serve as the 

infected snails in the study. It is not sure how many times a particular snail was used in 

the trials as it states that seven snails were used in the experiment. Also the mean number 

of cercariae released was measured for two days and the study was conducted for 10 

days. There is uncertainty in if this pre-measurment accurately reflects the true number 

of cercariae used in each of the trials given that the reviewer is unsure of how many times 

a snail was used in the trial and if they tend to shed a similar number of cercariae over the 

course of 10 days. An actual count of the number of cercariae used in each trial would 

have eliminated this uncertainty. However, it is possible that the tadpoles were exposed 

to different numbers of cercariae [or amount of chemical cue] each day and it is uncertain 

how that may have impacted behavior. 

The authors acknowledge the difficulty in standardizing comparisons between predator 

and parasite responses. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 
209

3.49. Rohr, J. R., T. M. Sesterhenn and C. Stieha. 2011. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Rohr, J. R., T. M. Sesterhenn and C. Stieha. 

2011. Will climate change reduce the effects of a pesticide on amphibians?: partitioning 

effects on exposure and susceptibility to contaminants. Global Change Biology. 17: 

657 - 666 (ECOTOX Ref. No. 153803) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/7/11 

Summary of Study Findings: 

The objective of this study was to test for an interaction between a temperature gradient 

and exposure to the herbicide atrazine on growth, survival, and hatching and metamophic 

timing of the streamside salamander (Ambystoma barbouri) isolating effects of 

temperature on both exposure and susceptibility (adverse effects from) to atrazine. 

Methods 

Wild collected salamander eggs (Harrison stage 18-28) were separated and then 12 eggs 

(embryos) each placed into 64 glass bowls containing 2 L of dechlorinated tap water. All 

bowls were aerated and all larvae were fed live blackworms (Lumbriculus variegatus) ad 

libitum. Bowls were divided equally (16) in four Percival environmental chambers. 

Technical grade atrazine (99% ai) was used; atrazine exposure concentration included 0, 

4, 40 and 400 μg/L. A solvent (acetone) control 165 mL/2 L (8.25%) was used; however, 

no negative control since [according to the authors] similar solvent concentrations were 

shown to have no significant effect. Study was conducted using static renewal with 

complete water changes once per week. 

EMBRYO STUDY

For the embryo studies, two environmental chambers were set at 13o

C and two were set 

at 19oC; each chamber had 4 replicates of each atrazine concentration (2 

chambers/temperature x 4 replicates/chamber x 4 treatments x 2 temperatures = 64 

bowls). The embryo study lasted until all embryos hatched or were dead (49 days) at 

which point all larvae were euthanized. Bowls were checked daily for hatching and 

mortality; larval mass was determined from formalin-preserved specimens 

LARVAL STUDIES

Larval studies were conducted using 4 replicates for each atrazine concentration within 

each of the 4 environmental chambers set at different temperatures: 16, 19, 22 and 25o

C. 
210

Two of the replicates of each atrazine concentration in each chamber were transferred to 

water containing acetone alone once the first larva metamorphosed, 19 days into the 

experiment (from the 25o

C chamber); therefore, half of the treatment replicates at each 

temperature had 19 days of atrazine exposure while the other half were exposed to the 

treatment concentration until they metamorphosed. Mortality and metamorphosis were 

quantified daily until all larvae metamorphosed or 

dieted (78 days from the start of the study) 

Results 

EMBRYO STUDY

According to the study authors, atrazine exposure 

significantly delayed hatching (Figure 1c; reproduced 

from report) and reduced larval mass (Figure 1d) at 

400 μg/L relative to the solvent controls. Exposure to 

atrazine at 4, 40 and 400 μg/L (log scale: 0.6, 1.6 and 

2.6 log μg/L) decreased embryonic survival (Figure 

1a). Atrazine exposure did not significantly affect 

larval survival (Figure 1b). Although the authors 

state that no significant interaction was detected 

between temperature and timing of hatching, 

embryonic survival or larval survival (Figure 1b); 

however, larval mass was significantly higher at 19 

than at 13o

C, the authors state that salamanders 

hatched sooner (Figure 1c) at 19oC. 

 
211

LARVAL STUDIES

According to the study authors, atrazine significantly delayed metamphosis (Figure 4a; 

reproduced from study) and reduced mass at metamorphosis (Figure 4b); temperature 

was positively associated with developmental rate (Figure 4a) but did not have a 

significant effect on size at metamorphosis (Figure 4b). The authors states that there was 

a "trend" for atrazine to be a "significant negative predictor of larval survival" and they 

site their previous studies showing that atrazine significantly reduced survival of larval A. 

barbouri and had one-tailed statistical test been run, the p value would have been 

significant. 

There was a significant interaction between temperature and duration for the slope of 

timing of metamorphosis (Figures 3b) and size at metamorphosis (Figure 3c); increasing 

temperature decreased the effect of atrazine on both timing of and size at metamorphosis 

[presumably by reducing exposure, rather than susceptibility to atrazine]. According to 

the authors, although atrazine had a greater [adverse] effect on growth at warmer than 

cooler temperatures when the duration of atrazine exposure was controlled (embryonic 

study) this increase in toxicity was small in comparison to the positive effects of 

increased temperature by accelerating the salamanders' development thereby reducing 

exposure to atrazine (larval study). 
212

The authors conclude that increasing atrazine concentrations reduced growth, delyaed 

hatching and metamorphosis, and decreased embryonic and larval survival and that with 

the exception of growth, increasing temperature did not enhance the toxicity of atrazine, 

but did generally ameliorate the adverse effects of atrazine by accelerating development 

and reducing the duration of atrazine exposure. 

Description of Use in Document (QUAL, QUAN, 

INV):

Invalid based on non-recommended solvent and solvent 

concentration 

Rationale for Use: 

The embryo study shows in general that there was much 

higher mortality of embryos and post-hatch larvae at 

19o

C. Embryos at 19 oC took less time to hatch (13 - 14 

days) and those reared at 13oC (27 - 31 days); in spite of 

the increased time to hatch, embryos had a lower mass at 

hatch compared to embryos reared at 19oC. 

The larval study demonstrates that increasing water 

temperature from 16 to 25 oC decreased the length of 

time to metamorphosis; although the increase in water 

temperature from 22 to 25o

C did not appear to markedly 

affect time to metamorphosis. Mass at metamorphsis 

appeared to fluctuate sporadically across the 

temperatures tested; however, in general, the reduced 

time to metamorphosis relative to other temperatures 

examined, at 25 oC appears to have been associated with reduced mass of the 

metamorphs. 

Limitations of Study: 
213

Wild-caught animals; therefore, prior exposure history is unknown. Eggs were separated; 

however, it is uncertain whether this involved de-jellying the eggs and how this 

separation effort may have influenced exposure. No negative control; authors assumed 

that the co-solvent would not affect study (based on historical data). Developmental 

stage of the test animals is not well characterized and it is unclear whether all of the test 

animals were at an identical stage of development. No error bars are provided in the 

report for embryo (Figure 1a) or larval survival (Figure 1b; therefore it is not possible to 

comment on the statistical significance of the figures. Poor larval survival across acetone 

control and atrazine treatments (roughly 60%; depicted in Figure 1b) suggests that 

survival was poor across all of the animals in the 19 oC treatment and as such, it may be 

difficult to make inferences about other treatment effects in this group. 

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
214

3.50. Spolyarich, N., R. Hyne, S. Wilson, C. Palmer and M. Byrne. 2010. 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Spolyarich, N., R. Hyne, S. Wilson, C. 

Palmer and M. Byrne. 2010. Growth, development and sex retios of Spotted Marsh Frog 

(Limnodynastes tasmaniensis) larvae exposed to atrazine and a herbicide mixture. 

Chemosphere 78: 807 - 813. (ECOTOX Ref. No. 153638) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/5/11 

Summary of Study Findings: 

The purpose of this study was to investigate the effects of atrazine, metolachlor and 

thiobencarb on the growth, development and sex ratio of developing spotted marsh frog 

which is indigenous to Australia. 

Methods 

Larvae were rear in 80-L aquaria filled with dechlorinated tap water with complete water 

renewal every 14 days. Larvae were fed every 2nd day with a 1:1 mix of Sera Micron®

and Nutrafin® Fry Food with increasing food ration as the animals matured. The larval 

development assay was based on Xenopus Metamorphosis Assay (XEMA) with exposure 

starting at early limb bud emergence (Gosner stage 28) and continuing until the majority 

of larvae were undergoing tail resorption (Gosner stage 42). Each replicate contained 10 

larvae in 10 L of water within 14.5-L glass aquaria fitted with a glass lid to limit 

evaporation. Each aquarium was aerated and was on a 12:12 light:dark cycle. Exposure 

commenced just prior to gonadal differentiation (Gosner stage 30). Animals were 

removed at Gosner stage 44 or 45. Spiking took place after every water renewal after 

every 3rd or 4th day of exposure. DO, temperature and pH were measured daily. Table 1

(reproduced from report) summarizes the various treatments. Sex ratios initially based on 

gross gonadal morphology. Male gonads from Tests 1, 6, 7 (atrazine only) and Test 4 

(atrazine, metolachlor and thiobencarb mixture) were examined histologically for the 

presence of oocytes. Atrazine (98% ai), metolachlor (99.5% ai), thiobencarb (99% ai) 

concentrations were analyzed weekly in the 4-day water following the water renewal. 
215

Results 

No atrazine, metolachlor or thiobencarb were detected in controls. Recovery of atrazine 

was on average 121.5%; average recovery of metolachlor was 90.8%; average recovery 

of thiobencarb was 34%. Thiobencarb recovery was 27.2% in a mixture with atrazine 

and 28.1% in a mixture with atrazine and metolachlor. 

Atrazine, metolachlor and thiobencarb had no observable effect on the development of 

the spotted marsh frog larvae at the concentrations tested either independently or in 

binary or ternary pesticide mixtures. Figure 1 (reproduced from report) dipicts mean 

Gosner stages in atrazine treatments showing that they did not vary from controls by 

more than one or two stages at any time period. No significant differences were found 

between the treatments and controls for any test. 

Atrazine, metolochlor and thiobencarb did not have a significant effect on total body 

length at the concentrations tested separately or in binary or ternary pesticide mixtures. 

Figure 2 (reproduced from report) shows mean lengths of larvae exposed to atrazine of 

0.1, 3 and 30 μg/L over 4 weeks compared to controls (Test 7). This authors noted that 

this particular test produced the largest although not significant difference in mean length 

of treated animals compared to controls with tail resorption occurring noticeably faster in 

the 0.1 μg/L atrazine treatment. This was not observed in other tests though. 
216

Mortality 2.3% (18/800 animals) died prior to the conclusion of the exposure period and 

mainly occurred during the first week of exposure; the authors attributed this to handling 

stress. There was no evidence of a concentration-dependent effect on mortality. "Kinky" 

tail was observed in 2% of the animals and was equally distributed across controls and 

treatments; crooked dorsal stripe was observed in 1.9% of the animals and was again 

randomly distributed among treatments and controls. 

Combining controls across tests, the sex ratio was 50.3:49.7 male:female. According to 

the authors, none of the sex ratios observed in the treatments were significantly different 

from controls (Table 2; reproduced from report). Female dominated ratos were observed 

in the 1 and 3 μg/L atrazine treatments of Test 1, but the authors note that this trend was 

not strong in the 3 μg/L atrazine treatment in Tests 6 and 7. 

Testicular ovarian follicles were observed in one male at Gosner stage 44 after 4 weeks 

of exposure to atrazine at 3 μg/L; the testes of this animal were "ambiguously" shaped. 

From the same treatment, another male at Gosner stage 41 had a enlarged area of the 

testes 
217

The authors conclude the exposure of spotted march frog lavae to atrazine (0.1, 1, 3 and 

30 μg/L, metolachlor (0.1, 1 and 10 μg/L) and thiobencarb (90, 180, and 360 μg/L) from 

Gosner stage 28 to 42 under controlled lab conditions had no significant effect on tadpole 

growth, development and sex ratio. Binary mixtures of atraine and thiobencarb as well as 

ternary mixtures of all three also had not significant effect on the developing larvae; 

abnormal gonad morphology was observed on two occassions, both from atrazine at 0.1 

μg/L with one larvae with testicular oovarian follicle but this did not exhibit a 

concentration associated response. 

 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: 

Study provides information on the effects of atrazine, metolachlor and thiobencarb both 

singularly and in combination on the spotted marsh frog. Studies of atrazine were 

reproduced three times with relatively consistent results for sex ratios. 

Limitations of Study: 

Wild-caught animals; therefore, prior exposure history is unknown. Histology was only 

conducted on male gonads. Sex was apparently based on gross gonadal morphology 

alone and was not verified across all of the test animals with histology. Given the n value 

(3), it is presumed that the graphs for mean rate of development (Figure 1) and mean 

length (Figure 2) represent pooled data from the three atrazine studies; it would have 

been informative to see the graphs for each atrazine study. It is unclear why similar 

graphs are not available for the various herbicide mixtures. In addition, based on the 

figures, it appears that psueoreplication occurred as the sample size for each treatment is 

30 (total individuals exposed per treatment) as oppose to ‘n’ of 3 (three replicates/trials 
218

for each treatment). Given that no effect was observed with presumably a sample size of 

30 (which greatly increases the power of the test), reducing the ‘n’ to 3 is expected to 

give the same result. 

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
219

3.51. Storrs, S.I. and J.M. Kiesecker. 2004 

OPEN LITERATURE REVIEW 

Chemical Name: Atrazine (Exposure of 4 species of tadpoles) 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 78290 

Storrs, S.I. and J.M. Kiesecker. 2004. Survivorship Patterns of Larval Amphibians 

Exposed to Low Concentrations of Atrazine. Environ. Health. Perspec. 112(10). p. 

1054-1057. 

Purpose of Review: Registration Review 

 

Date of Assessment: 4/20/12 (review based on review completed in 4/06) 

Brief Summary of Study Findings: 

Long-term exposure (~ 30 days) of atrazine (85.5%) ( nominal = 3, 30, or 100 ppb; 

measured = 2.84 + 0.05, 25.2 + 1.82, and 64.8 + 2.88 ppb) to four species of tadpole 

frogs including spring peepers (Pseudacris crucifer), American toads (Bufo americanus), 

green frogs (Rana clamitans) and wood frogs (Rana sylvatica) was studied at early 

(Gosner stages 25-27) and late (stages 29-36) developmental stages. The results of the 

study showed counterintuitive patterns of survivorship; survival was significantly lower 

for all animals exposed to 3 ppb compared with either 30 or 100 ppb, except the late 

stages of the toad and wood frog. There were significant differences in survivorship 

between the 3 ppb treatment group and the control for the late stage of the American toad 

and both developmental stages of the green frog. However, no significant differences in 

survivorship, relative to the control, were observed for late spring peepers, early 

American toads, and late wood frogs. The study author suggests that greater mortality at 

lower doses than higher doses is a pattern characteristic of many endocrine disruptors. 

Results show that animals exposed to low concentrations of atrazine died sooner that 

animals exposed to medium or high concentrations, indicative of a U-shaped curve. 

Methods: Treated water and food was replaced every 3 days. Experiments for each 

species were performed separately. Smaller animals (spring peepers, toads, and earlystage green frogs) were kept in 120 mL polypropylene cups in 100 mL dechlorinated 

water (w/treatments); larger animals (late wood and green frogs) kept in 750 mL highdensity polypropylene cups in 500 mL dechlor water. Photoperiod = 12 h light/dark; 

temp = 22 o

C. Animals fed crushed alfalfa every 3 days when water was changed. 

Description of Use in Document: Qual: 

 
220

Rationale for Use: Provides information on survival however, given results do not 

provide a clear and consistent response. 

Limitations of the Study: No raw data provided. Time to significant mortality (relative 

to the control) was not discussed. Animals that metamorphosed during the experiment 

were eliminated from the data set; therefore, sample size for each treatment for late-stage 

animals varied. The sample size for the late stage American toad was < 7. Statistical 

patterns may be influenced by differences in sampling sizes. A commercial preparation 

of atrazine containing 85.5% atrazine was used to create the treatment levels; the 

remaining 14.5% of this field-grade atrazine contains other chemicals (eg., surfactants) 

that may have an impact on survivorship patterns of larval amphibians. Survivorship 

patterns were presented as survival probability; therefore, it was not possible to determine 

or quantify the number of days until death or the overall mortality at the end of the 

experiment. 

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, 

Office of Pesticide Programs 

 
221

3.52. Storrs Méndez, S.I., D.E. Tillitt, T.A.G Rittenhouse, R.D. Semlitsch. 

2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Storrs Méndez, S.I., D.E. Tillitt, T.A.G 

Rittenhouse, R.D. Semlitsch. 2009. Behavioral response and kinetics of terrestrial 

atrazine exposure in American toads (Bufo americanus). Arch. Environ. Contam. 

Toxicol. 57: 590-597(ECOTOX Ref No. 118898). 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 3/3/12 

Summary of Study Findings: 

The objective of the study was to evaluate 1) if juvenile American toads avoid soils 

treated with atrazine; 2) if the toads can absorb atrazine across pelvic patch; and 3) if so, 

how is the atrazine distributed among the organs and eventually eliminated. 

Methods 

Terrestrial phase American toads (Bufo americanus) (reared from egg masses collected 

from Thomas S. Baskett Wildlife Area, Boone County, MO) were placed into test 

chambers that contained control soil as well as soil containing either 80 or 1430 μg/kg 

atrazine (99.2%). Atrazine stocks were prepared in acetone (5mL stock added to 800mL 

water before dosing soil). Chambers were oriented both north-south or east-west to 

control for directional bias. The toads were randomly assigned to 1 of 18 chambers on 

night 1 and 1 of 9 chambers on night 2. Both nights included all treatments, each toad 

used only once, and after toads were weighed they were acclimated to chambers and 

toads were observed with a red-light headlamp. Initial choice was recorded by noting the 

side the toad moved to after it left the release platform. Then at 3-minute intervals, it was 

noted what side the toad was on for the first hour and then every 12 hours for 2.5 days. 

For the uptake and elimination test, 17 toads from the soil study were used and were 

placed into 1-L glass jars that were tilted to allow for both a dry area and an area with 

treated water (460 μg/L atrazine). Food was withheld for 72 hours prior to initiation and 

toads were placed in empty test chamber overnight to allow dehydration. An attempt to 

empty bladders prior to test to obtain standard mass was made but 10 out of 17 animals 

did not empty bladder. Location of the pelvic patch was recorded during test. Toads 

were removed for analysis at 2, 4, 8, 12 and 24 hours after dosing. At 8 and 12 hours, 

any remaining treated water was removed and replaced with clean water to examine 
222

elimination. Organs were removed and measured for total radioactivity. Water samples 

were also analyzed. 

Results 

For the soil choice study, there was no significant choice of soil; while more toads were 

observed on atrazine treated soils, there was no pattern of significance. Measured 

atrazine concentrations in the soil were 59% lower than nominal concentrations. In the 

uptake and elimination study, 93% of the toads placed their pelvis in water at study 

initiation and radioactivity was removed from water in parallel to increase in body mass 

with an increase in radioactivity in water at between hour 4 and 8 and decrease after hour 

8 water change. The greatest radioactivity per organ was the intestines. According to the 

study authors, the greatest concentration per mg organ was in the gall bladder with peak 

concentrations in the gall bladder and intestines at hour 8 [Figure 4 indicates the peak 

was at hour 12 for both tissues]. Peak concentrations in other organs (i.e., liver, stomach, 

kidneys) was at hour 2. 

Description of Use in Document (QUAL, QUAN, INV): QUAL 

Rationale for Use: 

This study demonstates the movement of atrazine once it penetrates the skin of a toad. 

However, the endpoints evaluated here are not clearly or quantitatively linked to the 

endpoints of survival, growth and reproduction used in a risk assessment. While the 

study evaluated movement from atrazine treated water, statements about potential uptake 

while traversing atrazine contaminated soil cannot be made as the study evaluated a water 

only exposure and not a soil exposure. 

Limitations of Study: 

The toads used for the test were reared from eggs collected at a wildlife area, therefore, 

there is uncertainty in previous exposure to contaminants. Also, the type of material for 

the rearing and test chambers were not described as well as if the feed was free of 

potential contaminants. 

Measurements of atrazine in the soils were 59% lower than nominal. As these soil 

samples were stored for 5 months after the study was conducted, there is uncertainty in 

the actual exposure concentration as it is not known whether the decrease in nominal 

concentration was due to degradation during storage or not. 

According to the study, the atrazine stocks for the uptake study was prepared in ethanol. 

The concentration of the ethanol was 0.01% [reviewer calculated – 34 μL [0.034 mL] 

stock in 340mL dilution water]. To what extent the solvent influenced the uptake of the 

atrazine is not known. 

As 10 out of 17 animals did not relieve their bladders, the goal of trying to obtain a 

standard mass was not achieved for every animal and there is some uncertainty in how 

this affected subsequent mass analysis. 
223

According to the authors, one gall bladder burst during dissection at hour 2. It is 

uncertain in if it then further contaminated the other organs and according to the degrees 

of freedom in Table 1 and 2, it appears that it was included in the analysis. 

According to the study authors, 17 randomly selected toads from the soil choice test were 

used for the uptake and elimination study. As these toads appeared to be used in a 

previous experiment, and while the studies were 4 months apart, there is uncertainty in 

how exposure in the previous experiment may have affected the uptake study. 

Total radioactivity was measured in the uptake study with no further analysis to measure 

metabolites. Therefore, comparisons among organs and timepoints within an organ are 

based on the assumption that the radioactivity measured is parent atrazine when in fact it 

may be a combination of different metabolites and parent. 

Primary Reviewer: 

Amy Blankinship, Chemist, Environmental Risk Branch 3, Environmental Fate and 

Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

 

Not applicable 

 
224

3.53. Storrs-Méndez, S. I. and R. D. Semlitsch. 2009 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Storrs-Méndez, S. I. and R. D. Semlitsch. 

2009. Intersex Gonads in Frogs: Understanding the Time Course of Natural 

Development and Role of Endocrine Disruptors. Journal of Experimental Zoology (Mol 

Dev Evol) 312B: 1 -11. (ECOTOX Ref. No. 153841) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/2/00 

Summary of Study Findings: 

The objective of this study was to determine whether amphibians with an accelerated rate 

of sexual differentiation were more susceptible to endocrine disruption as larvae and 

metamorphs (juveniles) than species with slower rates of sexual differentiation. The goal 

of this study was to determine whether this susceptibility has an effect on gonad 

morphology in terrestrial anurans no longer exposed to aquatic contaminants. To do this, 

the authors examined gonadal morphology over time in solvent (ethanol) control, positive 

control (estradiol) and atrazine treatments in three species exhibiting differing rates of 

ovary differentiation. Of the species tested, American toads (Bufo americanus) have the 

shortest larval period and rates of differentiation; gray treefrogs (Hyla versicolor) have 

intermediate larval period and a “retarded rate of differentiation; the Southern leopard 

frog (Rana sphenocephala) have long larval periods and have an accelerated rate of ovary 

development. 

Methods 

Larval studies examined three atrazine concentrations (1, 3 and 30 μg/L), ethanol control 

and a 17-β estradiol (10-7M) control. The study was conducted using static-renewal (72-

hr) conditions. Exposure was initiated at Gosner stage 25. For B. americanus: 5 

treatments replicated 10 times in three blocks; H. versicolor: 5 treatments replicated 6 

times in 5 blocks and R. sphenocephala: 5 treatments replicated 16 times in three blocks. 

Blocks represented different shelves on which the treatment units were located in the 

laboratory. Each replicate consisted of 1 tadpole in 1-L jars filled with 800 mL water. 

Animals with a least one forelimb (Gosner stage 42 - 46) were removed from the test 

chambers and allowed to fully metamorphose in plastic containers with at least 1 cm of 

water and a dry area. Ten animals from each treatment were removed at 3-wk intervals; 

any animals remaining at completion of metamorphosis (complete tail resorption [Gosner 

stage 46]) were sacrificed for assessment of gonadal histology to assess the proportion of 
225

males, females, underdeveloped testes UT), testicular oocytes (TO; testes with 0 – 30% 

oocytes) and ovotestes (OVT; testes with >30% oocytes). R. sphenocephala sacrificed at 

3, 6 and 9 weeks and at completion of metamorphosis; H. versicolor and B. americanus

sacrificed at 1 3 weeks and at metamorphosis. For those animals that completed 

metamorphosis, body weight and time to metamorphosis were recorded. For all of the 

test animals, the kidney-gonadal complex was prepared for histological analysis. Studies 

with B. americanus were conducted 1 year after the studies with the other two species. 

 Juvenile studies examined two atrazine concentrations (1 and 10 μg/L), solvent (ethanol) 

control, and 17-β estradiol control. According the study methods, 20 tadpoles in 20 L of 

water for Hyla and Rana and 12 tadpoles/20 L of water for Bufo. The study was 

replicated 3 times for the 4 treatments with one replicate per vertical block. Animals 

were reared [in pens] to 4 months for Rana and Hyla; Bufo were reared [in pens] to 

“nearly” 6 months of age. The kidney-gonadal complex of the test animals were 

prepared for histological analysis. 

Results 

Treefrogs were raised for 124 ± 0.15 (mean± std err) days after larval exposure started; 

leopard frogs were raised for 120 ± 0.05 days and toads were for 166 ± 1 days after larval 

exposure was initiated. Measured concentration of atrazine recorded in the study are 

reported in Table 1. Studies with treefrogs and leopard frogs were conducted in 

Table 1. Summary of measured exposure concentrations of atrazine for larval 

studies. 

Test Species 

 Nominal Atrazine Concentrations 

1 μg/L 

Treatment 

3 μg/L 

Treatment 

30 μg/L 

Treatment 

B. americana 2.7 ± 0.75 7.55 ± 2.82 124.87 ± 41.26 

H. versicolor 0.92 ± 0.07 2.81 ± 0.15 25.1 ± 7.06 

R. sphenocephala 0.80 ± 0.05 3.13 ± 0.25 30.36 ± 1.23 

Table 2. Summary of measured exposure concentrations of atrazine for juvenile 

studies. 

Test Species 

 Nominal Atrazine Concentrations 

1 μg/L 

Treatment 

10 μg/L 

Treatment 

B. americana 3.02 ± 0.25 31.90 ± 1.79 

H. versicolor 4.12 ±0.10 29.52 ±8.74 

R. sphenocephala 1.46 ± 0.11 24.58 ± 9.60 

Figure 1 (reproduced from the study) depicts the sex ratios of B. americanus (A) and H. 

versicolor (B) at metamorphosis and shows that the percentage of females was 

significantly higher in toads treated with atrazine 30 μg/L compared to controls and that 
226

in Hyla, sex ratios were significantly different than controls treefrogs treated with 

atrazine 1 and 30 μg/L but not at 3 μg/L. Sex ratios of ethanol control toad did not differ 

significantly from estradiol controls (Figure 1A); however, for treefrogs, sex ratios in the 

ethanol control were significantly different (lower) than the estradiol control 

Figure 2 (reproduced from the study) depicts sex ratios for R. sphenocephala after 6 wks 

(A), 9 wks (B) and at metamorphosis (C). There were no significant differences in sex 

ratio at 6 wks relative to the ethanol control; at 9 wks there were significant differences in 

the sex ratio of frogs treated with atrazine at 3 and 30 μg/L (but not at 1 μg/L) relative to 

the ethanol control; by metamorphosis there was no significant difference between 

atrazine-treated and ethanol control; however, sex ratio of frogs treated with 30 μg/L 

were significantly different than the estradiol control where the atrazine-treated frogs had 

a significantly lower number of females (roughly 27%) compared to estradiol-treated 

frogs with roughly 50% based on Figure 2C. For leopard frogs, the sex ratio of ethanol 

controls was only significantly different than estradiol controls at 6 wks (Figure 2B) 
227

Figure 3 (reproduced from the report) depicts the sex ratios for juvenile B. americanus

(A), Hyla versicolor (B) and R. sphenocephala (C). Although the figure caption reports 

that three concentrations of atrazine (1, 3 and 30 μg/L) were tested, the bar graphs 

correctly depict only two atrazine treatments (1 and 10 μg/L). 

The authors conclude that a phase of intersex 

gonads (TO or OVT) is normal during leopard 

frog sexual development, a species 

representing an accelerated rate of 

differentiation. Additionally, intersex gonads 

were found in juvenile stages of toads and 

treefrogs representing animals with slow and 

basic rates of gonadal differentiation, 

respectively, indicating that neither had 

completed sexual differentiation. The authors 

note that previous studies have shown 

inconsistencies and the absence of a clear doseresponse relationship among atrazine-treated 

amphibians and that longitudinal studies 

examining a broader range of development 

time are needed. The authors note that at 9 

weeks and at metamorphosis, Hyla and Rana

both had sex ratios that differed from controls 

and that both had higher incidences of females 

and abnormal gonads than [ethanol] controls 

and were similar to those treated with estradiol. 

They note that underdeveloped testes (UT) 

were found only in animals exposed to a 

chemical treatment: Hyla had on UT at the 1 

μg/L atrazine at metamorphosis and 2 among 

estradiol exposed animals in the juvenile study 

while Rana had one in each of the atrazine 

treatment concentrations in the study of 

juvenile amphibians. The authors conclude 

that UT are not "normal" since they are not found in [ethanol] controls. According to the 

authors, R. sphenocephala would serve as a good model for examining the effects of 

contaminants in gonadal development since the frog has long larval periods to ensure 

exposure and the longest period of gonadal development and because they reach full 

gondal development the fastest [of the species tested]. 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: 
228

Although there are a number of uncertainties associated with the study, it provides useful 

information toward understanding the range of gonadal developmental effects reported in 

previous studies. 

Limitations of Study: 

Parental animals were wild-caught and their prior exposure history is unknonw. No 

negative control was run. Source of the dilution water not stated. Although not clearly 

articulated, the larval studies appear to have been conducted in glass containers until 

larvae were close to metamorphosis when the animals were then transferred to plastic 

containers. The use of plastic containers is discouraged particularly in studies examining 

potential developmental effects. 

It’s unclear from the discussion of the experimental design how the total number of 

replicates (reps x blocks) relate to the n values cited for each of the species: B. 

americanus (10 reps x 3 blocks=30 units compared to n=150), H. versicolor (6 reps x 5 

blocks=30 units compared to n=150), and R. sphenocephala (16 reps x 3 blocks= 48 

units compared to n=240). This implies that for each replicate there are 5 animals; 

however, the methods section states that each animal was housed separately. 

It's not entirely clear from the methods section how juveniles were exposed to atrazine, 

i.e., whether exposure was just during their larval develop period or whether they were 

somehow exposed during the juvenile period as well. 

The authors do not report whether mortality was an issue in the study. 

The limit of detection (LOD) for atrazine and estradiol are not reported; the limit of 

quantification (LOQ) for atrazine is 0.05 μg/L. In the B. americanus study, atrazine was 

detected in the estradiol control at 0.05 ppb. Measured concentrations in the 1, 3 and 30 

μg/L treatments with larvae were 2.7, 2.5 and 4.2X the nominal concentrations while for 

the other two test species, measured concentrations were relatively consistent with 

nominal. For the studies with juveniles, measured concentrations in the 1 and 10 μg/L 

groups were roughly 3X nominal for B. americana, 3 – 4X for H. versicolor and 1.5 – 

2.5X for R. sphenocephala. These differences were attributed to evaporation of the 

dilution water; however, a considerable amount of water must have evaporated from the 

treatment containers. Although exposure to atrazine was conducted throughout the larval 

study period, it is unclear when measured concentrations were recorded. 

The authors report that kidney-gonadal complex was sectioned longitudinally into 7 μm 

sections; however, it is not stated whether the entire complex was sectioned. 

The sex ratio at metamorphosis of control H. versicolor depicted in Figure 1 is roughly 

27% female and appears to be relatively low. The sex ratios of R. sphenocephala

ethanol controls sampled at 6 (Figure 2A) and 9 weeks (Figure 2B), were roughly 78% 

and 75% female, respectively, and appear to be relatively high. The sex ratios of the 

ethanol controls then drop to roughly 29% female by metamorphosis (Figure 2C). The 

changing sex ratios suggest that gonadal development was inconclusive prior to 

metamorphosis and does not serve as reliable/decisive indicator of sex. 
229

Sex ratios in the estradiol controls were only significantly different from ethanol controls 

in the treefrogs at metamorphosis (Figure 1B) and in the leopard frogs at 6 wks of 

exposure (Figure 2B); however, in the leopard frogs the number of females was reduced 

relative to the ethanol control whereas in the toad, the number of females was higher [as 

expected]. 

Figure 5D of the paper (not reproduced in this DER) reports results from 10 μg/L 

atrazine treatments form both larval and juvenile experiments; however, larvae were not 

exposed to 10 μg/L. 

The authors note that the number of testicular oocytes were higher near the proximal end 

of the testes of toads where the Bidder’s organ is located and appeared to be a “bleeding” 

into the testicular tissue from the Bidder’s organ. Although the Bidder’s organ is 

characterized as a “latent” ovary and occurs in both males and females, its function and 

extent of development is generally not well characterized and it is unclear whether the 

Bidder tissue is actually infiltrating (bleeding into) the testes, whether this is an artifact 

from sectioning for histology, or whether this is an actual effect from exposure to 

atrazine. 

The authors' observation that underdeveloped testes (UT) represented an "abnormal" 

condition did not appear to be a consistent means of detecting treatment effects since it 

appeared infrequently among atrazine- and estradiol-treated amphibians. Also, UT did 

not appear to be dose-responsive nor did it consistently appear in the estradiol-treated 

animals. 

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 
230

3.54. Wan M.T., C. Buday, G. Schroeder, J. Kuo, and J. Pasternak. 2006 

Chemical Name: Atrazine 

CAS No: 1912-24-9 

ECOTOX Reference Number and Citation: 89626 

Wan M.T., C. Buday, G. Schroeder, J. Kuo, and J. Pasternak. 2006. Toxicity to Daphnia 

magna, Hyalella azteca, Oncorhynchus kisutch, Oncorhynchus mykiss, Oncorhynchus 

tshawytscha, and Rana catesbeiana of Atrazine, Metolachlor, Simazine, and Their 

Formulated Products. Bull. Environ. Contam. Toxicol. 76: 52-58 

Purpose of Review: Registration Review

Date of Assessment: 5/1/12 

Brief Summary of Study Findings: 

Technical and an atrazine formulation (Atrazine 500 (48.5%) were used to in acute 

toxicity tests with several species. Only atrazine is discussed in this review. 

The test conditions are provided in the table below (reprint from the study). 

The acute toxicity for technical and atrazine formulation are provided in the table below 

(reprint from the report). 
231

A 28-d Hyalella azteca survival study in sediment was also conducted and the results are 

presented below (reprint from study) 

Description of Use in Document: Qualitative. 

Rationale for Use: Provides information about effects from atrazine interaction but 

there are uncertainties in study. 

Limitations of the Study: 

Solvent concentration not reported; raw data not provided, control mortality not reported 

 

Reviewer: Amy Blankinship, ERB3 
232

3.55. Williams, B. K. and R. D. Semlitsch. 2010 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Williams, B. K. and R. D. Semlitsch. 2010. 

Larval Responses of Three Midwestern Anurans to Chronic, Low-Dose Exposures of 

Four Herbicides. Arch. Environ. Contam. Toxicol. 58: 819 - 827 (ECOTOX Ref. No. 

153825) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/5/11 

Summary of Study Findings: 

The objective of this study was to expose three common species of tadpoles to 

conservative levels of atrazine, S-metolachlor, and glyphosate end-use herbicides 

formulations throughout the larval period to test for survival differences or life-history 

trait alterations. 

Methods 

Eggs collected from the field and maintained at 25oC until hatch; Gosner stage 25 larvae 

randomly assigned to individual 1-L glass jars. Species included American toads (Bufo 

americanus, western chorus frog (Pseudacris triseriata) and gray treefrogs (Hyla 

versicolor). Larvae were exposed to nominal concentrations of formualted atrazine 

(Atrazine® 4L; 48.8% ai; 0.2 and 3 μg a.i./L), S-metolachlor (Dual® II Magnum; 82.4%; 

0.2 and 10 μg a.i./L), and two formulations i.e, Roundup® Original Max (48.7%) and 

Roundup® WeatherMax (48.8%) containing glyphosate potassium salts (each at 0.5 and 

572 μg a.e./L. There were 10 replicates per treatment under static renewal (72-hr) 

conditions; chorus frog and toad larvae fed Tetramin® fish flakes and ground rabbit chow 

(50:50) after each water change; treefrogs fed Tetramin® alone. Survival, mass at 

metamorphosis, length of larval period were measured. Metamorphosis was defined as 

the emergence of at least one forelimb (Gosner stage 42) at which time the animal was 

transferred to a plastic container until it had fully metamorphosed (complete tail 

resorption; Gosner stage 46) which typically took an average of 3 days. 

Results 

Mean-measured concentrations (over three time points in the study) of atrazine in the low 

and high treatments were 0.28 and 1.25 μg a.i./L, respectively. Atrazine in controls was 

less than 0.02 μg a.i./L. All of the toads and chorus frog survivors metamorphosed; 

whereas 26% of the surviving treefrogs had metamorphosed by the end of the study. 
233

Only 20% of the chorus frogs exposed to Roundup® WeatherMax survived through 

metamorphosis (Figure 1; reproduced from study). Survival of toads and treefrogs was 

unaffected by the herbicide exposure (Figure 1). Larval period for toads was 

significantly longer larvae exposed to glyphosate at 572 μg a.e./L; larvae exposed to 

Roundup® WeatherMax metamorphosed 14% later (27.8 days) while larvae exposed to 

Roundup® Original Max metamorphosed 8% later (26.2 days) compared to controls (24.3 

days) (Figure 2; reproduced from study). Exposure of chorus frogs to Roundup®

WeatherMax at 572 μg a.e./L had a marginally significant effect on time to 

metamorphosis, i.e., 13% longer (32.0 days); however, the high levels of mortality in this 

group only left two animals to complete metamorphosis. Larval periods for treefrogs 

were not affected by herbicide treatments (Figure 2). Mass at metamorphosis did not 

differ with herbicide treatments for any of the species tested; the authors note though that 

the power of the study to detect effects on weight was low. 
234

The authors concluded that exposure to the glyphosate [formulated] product Roundup 

WeatherMax at 572 μg a.e./L resulted in 80% mortality of western chorus frog larvae 

that was likely the result of a "unique surfactant" formulation and that exposure to either 

formulation of glyphosate at 572 μg a.e./L lengthened the larval period for American 

toads. Chronic atrazine and S-metolachlor exposure induced no significant negative 

effects on survival, mass at metamorphosis, or larval period length at the levels tested. 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: 

Although there are a number of uncertainties associated with the study, it provides useful 

information toward understanding the range of gonadal developmental effects reported in 

previous studies. 

Limitations of Study: 

Wild-caught test animals; therefore, prior exposure history is unknown. Source of the 

dilution water not stated. Concentrations of glyphosate and S-metolachlor were not 

measured and are based on nominal concentration only. Control survival (roughly 80%) 

was relatively low for chorus frogs. 
235

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 
236

3.56. Zaya, R. M., Z. Amini, A. S. Whitaker, S. Kohler and C. F. Ide. 2011 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Zaya, R. M., Z. Amini, A. S. Whitaker, S. 

Kohler and C. F. Ide. 2011. Atrazine exposure affects growth, body condition and liver 

health in Xenopus laevis tadpoles. Aquatic Toxicology 104: 243 – 253. (ECOTOX Ref. 

No. 153847) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/19/11 

Summary of Study Findings: 

The objective of this study was to establish a standardized protocol to reproduce effects 

(i.e., increased mortality, reduction in metamorphic rate, and a reduction in body weight 

and length and fat body size) noted in a previous study and to augment these with data 

with additional assay to understand what is happening at the organ level. The second 

objective of this study was to begin to assess the cellular mechanisms these phenotypic 

effects in exposed African clawed frog (X. laevis) larvae. 

Methods 

Six separate experiments were conducted: 4 studies (A, B, C and D) with atrazine at 0 

and 400 μg/L; 1 study (Study E) with atrazine at 0, 25 and 200; 1 study (Study F) with 

atrazine at 0, 200 and 400 μg/L. Two lots of atrazine were used in the study and a stock 

solution (25 mg/L) was prepared by sonicating and warming (at 50o

C for no more than 15 

min). Five days post hatch (NF stage 47 larvae separated into bowls containing 1 L of 

spring water with and without atrazine: (6 larvae in Studies D and E), 8 (Studies A and 

C), 10 (Study F) and 22 (Study B); after 2 weeks, bowls had a final density of no more 

than 4 larvae per bowl and the spring water was replaced with dechlorinated tap water 

with and without atrazine for the remainder of the study. Bowls were stacked on 

laboratory shelves and relied on room temperature (19 – 23o

C) and ambient light. 

Exposure water was completely replaced three times per week. Larvae were fed 

powdered Purina rabbit chow (#5354) ad libitum. All larvae in each bowl were 

collectively weighed once per week during exposure until study termination and during 

this process each individual tadpole was staged. Appetite was monitored in three studies 

between 8 – 8:30 am 3 to 5 days per week by observing behavior pattern. Depending on 

the experiment, larvae were sacrificed at either NF stage 58 or 62 at which time animals 

were weighted, snout-vent length measured and age (in days) recorded. Gender was 

determined by gross [gonadal] morphology and the liver was removed and weighed. Fat 
237

bodies were photographed. Livers and fat bodies were then prepared for histology. 

Differential staining for glycogen and caspase-3 

Results 

Measured concentrations of atrazine in the he stock and 1 μg/L concentrations were 

within ±4% of nominal; however, neither of these represents actual treatment 

concentrations. According to the study authors, none of the animal exposed to atrazine at 

25 μg/L were significantly differenct from controls in terms of body weight. Body 

weights were sporadically different from controls in animals exposed to 200 μg/L; 

however, in all five of the experiments, larvae exposed to 400 μg/L had significantly 

(p<0.04) lower body weights than controls starting at 3 weeks of exposure (Figure 1; 

reproduced from report) . In studies C and F, larvae were collected early in exposure and 

weighed individually and exhibited significantly (p<0.03) lower body weights within 72 

hr of exposure. 

According to the study authors, control 

tadpoles developed at rates similar to 

those listed by Nieuwkoop and Faber 

and there were no differences in 

metamorphic stages between controls 

and larvae treated with atrazine at 25 and 

200 μg/L nor was there a difference in 

age at NF Stage 62. Changes in weekly 

metamorphic stage were inconsistently 

noted at 400 μg/L; studies C and D 

showed significantly lower (p<0.003) 

metamorphic stages. Although not 

measured in study C, study D, larvae 

were significantly (p<0.01) older than 

controls by 8% at NF stage 62 indicating 

a delay in metamorphosis (Figure 2; 

reproduced from report). 

There was no significant changes in 

feeding behavior at any of the [atrazine] 

exposure levels; however, for larvae 

exposed to atrazine at 400 μg/L feeding 

behavior was significantly increased 

during the second week of exposure in 

studies D (p<0.004) and F (p<0.02) 

(Figure 3; reproduced from report). 

There were no statistically significant 

differences in mortality between 
238

controls and atrazine-treated animals across the various studies (Table 1). 

Table 1. Summary of mortality rates in studies A through F 

Study A* Study B** Study D* Study E* Study F*

C 400 

μg/L C 400 μg/L C 400 μg/L C 200 μg/L C 400 μg/L 

Mortality 6% 7% 3% 0 5% 11% 2% 12% 1% 3% 

*

study conducted to NF Stage 62 

**study conducted to NF Stage 58 

There were no atrazine-related differences in sex ratio based on gross gonadal 

morphology compared to controls. There were no intersex animals noted in any of the 

atrazine-exposed animals; however, histology was not conducted. 

There were no statistically significant changes in body weight or snout-to-vent length in 

tadpoles exposed to 25 μg/L. In only one study (Study F) of two conducted using 200 

μg/L was body weight significantly (p=0.02) lower (15%) than controls. In terms of 

length, none of the larvae exposed to 200 μg/L were statistically different from controls. 

In all five studies using 400 μg/L, larvae weighed significantly (p≤0.05) less than 

controls by 13 – 22%; however, only animals from Study D were significantly (p≤0.01) 

shorter (6%) than controls. 

For fat bodies, none of the larvae exposed to 25 μg/L were significantly different from 

controls; however, larvae exposed to 200 and 400 μg/L had fat bodies that were 

significantly (p≤0.05) different (24 – 81% decrease in % body weight) from controls. 

There were no significant differences in relative liver weights (% body weight) in larvae 

exposed to 25 μg/L compared to controls. In one (Study F) of the two studies conducted 

at 200 μg/L was relative liver weight significantly (p≤0.05) lower (14%) than controls. 

In two (Studies D and F) of the four studies conducted at 400 μg/L were relative liver 

weights significantly (p≤0.05) different (14 and 20% lower, respectively) from controls. 
239

No “pathological” changes were observed in fat body and liver histology at any of the 

atrazine concentrations tested. There were no differences in liver glycogen or lipid 

content between atrazine-treated and control larvae; there was no difference in fat body 

lipid content between atrazine and control larvae. 

According to the study authors, liver sections from tadpoles exposed to 400 μg/L in 

Study D had significantly (p≤0.01) increased number of activated caspase-3 

immunopositive cells relative to controls and that these activated cells would likely 

become apoptotic and die.

The authors conclude that the NOEC from their studies is 25 μg/L, based on effects 

observed at the 200 and 400 μg/L treatments. These effects included reduced liver and 

fat body sizes and that the changes indicated that exposure to atrazine throughout larval 

development 

“compromised” the larvae and potentially decreased their ability to survive the stresses of 

metamorphosis or reduce reproductive fitness since frogs rely on stored lipids for these 

purposes. 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: 

Although the concentrations tested in this study are of uncertain ecological relevance and 

although the studies were separate, the studies provide information that mortality and 

gonadal development (based on gross morphology) were not affected at atrazine 

concentrations up to 400 μg/L. The statistically significant effects (reductions) on body 

weight and on fat body and liver weights expressed as percent body weight did not 

appear to be dose-responsive and did not appear to result in any abnormal histological 

effects on the organs. The activation of caspase-3 in hepatocytes of larvae from one of 

the four studies examining exposure to 400 μg/L is uncertain since none of the other 

studies replicated this result. 

Limitations of Study: 

Source of X. laevis eggs not stated; presumed to be Nasco. Stocking density of larvae 

ranged from 6 – 22/L; ideally, stocking density should be 1 larva/L. Relatively high 

loading rates coupled with feeding powdered rabbit chow ad libitum under 72-hr static 

renewal conditions could result in unfavorable water quality conditions. Also, reported 

environmental conditions suggest that water temperatures fluctuated broadly (19 – 23o

C) 

and that lighting was not uniformly distributed across the treatment units. Removing all 

of the larvae from each exposure to record collective weights is likely a major source of 

stress for the test animals and it is uncertain how accurate such weight measurements 

would be given the variable amount of water that may have been retained in the process. 

Although not discussed in the methods section, the results section suggests that several of 

the studies measured individual weights of the test animals; collective weight 
240

measurements would be stressful, but individual weight measurements would likely have 

increased the amount of handling and consequently stress. Staging each tadpole during 

the time they were removed from the tank further manipulated the animals and likely 

added to their stress level. Given that food was provided ad libitum, it is unclear how 

“appetite” could have been accurately assessed. Presumably the observation period, i.e., 

0800 – 0830 hrs, coincided with when food was added to each of the treatment units. 

The methods do not state how exactly the appetite measurement was “scored”, i.e., 

whether it was presence/absence or some gradient of response. 

While atrazine residues of 200 - 400 μg/L may be detected in puddles and ephemeral 

pools located in treated fields, these do not reflect values typically reported in surface 

water adjacent to field treated with atrazine. 

It sounds as though NF Stage 47 was selected as this would correspond to when Xenopus 

laevis larvae would initiate feeding. Exposure proceeded to NF Stage 62; this would 

correspond to metamorphosis when it is likely that test animals would cease feeding and 

begin to resorb tissues to sustain the metamorphosis process. As such, catabolism of fats 

and proteins is likely. By NF Stage 61 the filter apparatus of X. laevis larvae starts to 

degenerate and at Stage 62, the entire filter apparatus shows signs of cytolysis. In one 

study (Study C) tadpoles were collected after acute exposure to 400 μg/L; however, since 

exposures were conducted for several weeks, they could not be considered “acute”. The 

authors report "near-significant" results; however, such observations have little utility as 

there is no way to determine short of additional samples whether the effect is by chance 

alone. 

Only one study showed statistically significant (15%) reduction in body weight in larvae 

exposed to 200 μg/L but none of the larvae were significantly shorter. Although all of 

the animals exposed to 400 μg/L weighed significantly less (13 – 22%) than control, only 

animals from one of the five studies were shorter (6%). 

Although larvae exposed to either 200 or 400 μg/L had significant reduced fat body size 

(% of body weight; Table 2), the measurement endpoint did not appear to be dose 

responsive. In the one study (Study F) where exposure to both concentrations was 

examined, the reduction was relatively similar (28 and 31%). The highest reduction 

(81% in Study B) in the relative weight of fat bodies was observed in the 400 μg/L 

treatment; however, none of the other studies resulted in a similar magnitude response. 

Table 2. Summary of decrease in fat body size as a percentage of body weight. 

Study A* Study B** Study D* Study E* Study F*

400 μg/L 400 μg/L 400 μg/L 200 μg/L 200 μg/L 400 μg/L 

Fat Body 33% 81% 44% 24% 28% 31% 

Relative liver weight was also a variable measurement endpoint; in one to the two studies 

conducted at 200 μg/L and half of the studies conducted at 400 μg/L was the hepatic 

somatic index reduced. Again, the percent reductions at 200 and 400 μg/L do not appear 

to be substantially different. 

The only statistically significant measurement endpoint from histology was that larvae 

exposed to 400 μg/L had liver sections with significantly grated number of activated 
241

caspase-3 hepatocyes. The fact that this finding is limited to only one of the four studies 

which examined this exposure concentration limits its utility especially given that no 

other abnormal effects were reported through histology. 

The authors’ conclusions that exposure to atrazine throughout larval development 

“compromised” the larvae and potentially decreased their ability to survive the stresses of 

metamorphosis or reduce reproductive fitness is speculative and cannot be supported by 

the data contained in their study. Although the researchers believe that significant 

activation of caspase-3 that would likely lead to cellular apoptosis, histology did not 

reveal any liver pathology in atrazine-treated animals relative to controls. 

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
242

3.57. Zaya, R. M., Z. Amini, A. S. Whitaker, and C. F. Ide. 2011 

Open Literature Review Summary 

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Number and Citation: Zaya, R. M., Z. Amini, A. S. Whitaker, and 

C. F. Ide. 2011. Exposure to atrazine affects the expression of key genes in metabolic 

pathways integral to energy homeostasis in Xenopus laevis tadpoles. Aquatic Toxicology 

104: 254 – 262. (ECOTOX Ref. No. 153848) 

Purpose of Review (Note: DP Barcode required for Quantitative studies): 

Determine the relevancy of the study for evaluating the potential effects of atrazine on 

non-target organism, specifically amphibians. 

Date of Review: 11/19/11 

Summary of Study Findings: 

The objective of this study was to test the hypothesis that atrazine exposure altered 

energy homeostatsis in Xenopus laevis tadpoles. The authors relied on an reanalysis of 

Affymetrix microarray data generated in Langerveld et al., 2009 and included 

quantitative real-time polymerase chain reaction (qRT-PCR), ATP and ADP:ATP ratios 

to measure perturbations in energy homeostasis. 

Methods 

Animals used in this study were randomly selected from Study C and Study F reported in 

Zaya et al. 201177 (ECOTOX Ref. No. 153847; DER completed 11/19/11). Tadpoles 

were obtained from in-house breeding colony. Larvae (5-day post hatch; NF stage 47) 

stocked at a rate of 8 larvae/L until two weeks into the study and thereafter at 4 larvae/L. 

Study C consisted of 20 bowls/treatment and study F consisted of 20 control bowls, 10 

bowls at 200 μg/L and 20 bowls at 400 μg/L; each bowl contained 1 L of water and were 

maintained at 19 – 23o

C with ambient light exposure; water was completely replaced 3 

times weekly and animals were fed a suspension of powdered Purina rabbit chow (#5354) 

ad libitum. Exposure to 0, 200 or 400 μg/L continued from Day 5 post-hatch (NF stage 

47) for approximately 50 days until NF stage 62. 

GeneSifterTM, an on-line gene analysis software tool was used to determine which 

metabolic processes were “over” or “under-represented” in the overall gene expression 

observed through the Affymetric microarray analysis reported in Langerveld et al. 2009. 

Twelve chp files from control and 11 chp files from the 400 μg/L exposed larvae were 

 

77 Zaya, R. M., Z. Amini, A. S. Whitaker, S. Kohler and C. F. Ide. Atrazine exposure affects growth, body 

condition and liver health in Xenopus laevis tadpoles. Aquatic Toxicology 104: 243 – 253. (ECOTOX 

Ref. No. 153847) 
243

generated using Affymetrix Microarray Suite (version 5) software. Only changes that 

were statistically significant (p≤0.05) were accepted. 

For measuring differential gene expression a single tadpole from control and atrazine 

exposure bowls were collected at 24, 48, 72 hrs and at 14 days after exposure initiation. 

Total RNA was isolated from whole tadpoles of qRT-PCR assays; yeast mRNA was used 

as an internal control for the extraction and analysis. Genes of interest were selected 

because they were significantly changed (increased or decreased) based on the 

microarray analysis and they encoded what were considered rate-limiting enzymes in 

energy homeostatsis. Each qRT-PCR reaction (n=8 for both 0 and 400 μg/L) was run in 

duplicate. The mean value from each duplicate was calculated and only those duplicates 

with a standard deviation ≤0.25 were used for data analysis; gene expression was 

normalized to the internal yeast standard. 

Larvae from Study F were collected at 24 (control=10; 200 μg/L=9; 400 μg/L=11), 48 

(n=10 for all groups) and 72 hr (control=11; atrazine=10) of exposure); additionally, one 

tadpole from each bowl was collected at NF stage 62 (control=12; atrazine=8) and then 

weighted, length measured and its liver excised for ATP and ADP analysis. A known 

amount of ATP was spiked into a sample aliquot to determine recovery; recovery of ATP 

was >98% and it was assumed that there was a similar recovery for ADP. Firefly 

luciferase bioluminescence assay was used to assay ATP and ADP (where all ADP was 

first converted to ATP and total ATP was determined); ATP alone was measured using 

an ATP Determination Kit. The ratio of ADP:ATP could then be determined. 

Results 

Based on the Affymetric/GeneSifter analysis, up-regulated genes included those 

associated with the synthesis and degradation of ketone bodies, the urea cycle, regulation 

of autophagy, citric acid cycle, glutathione metabolism and metabolism of a number of 

amino acids (methionine, alanine, aspartate, arginine, proline, glycine, serine, and 

threonine. Down-regulation included glycolysis/gluconeogenesis, fatty acid elongation in 

mitrochondria and ribosomal proteins. 

The authors note (based on their previous publication) that larvae collected from Studies 

C and F were significantly (p≤0.03) smaller within 72 hr of exposure and at NF stage 62 

and had depleted fat stores at the end of exposure. 

Larvae from Study C were collected at 24, 48, 72 hrs and 14 days after initiating 

exposure and six genes were examined: hypoxia inducible fate 1α, carbamoyl-phosphate 

synthetase 1, E3 component of pyruvate dehydrogenase, acyl-CoA dehydrogenase family 

member 10, and 

glucocorticoid receptor protein. Of these, there was a significant decrease in gene 

expression of 

acyl-CoA dehydrogenase (p=0.03) and glucocorticoid receptor (p=0.04) at 24 hrs. Gene 

expression of the remaining genes and time points was no different than controls. 

Larvae from Study F were collected at 24, 48 and 72 hr after initiating exposure and one 

tadpole from each bowl was collected at NF stage 62. There were no significant 

differences in absolute ATP levels between atrazine-treated and controls within the first 

72 hrs of exposure nor in larvae tested at NF stage 62. The ADP:ATP ratio was not 
244

different between atrazine-treated and control animals at 24 – 72hr samplings. The 

authors report a near significant increase in ADP:ATP ratios in tadpoles exposed to 200 

μg/L at NF stage 62. 

The authors note that measurable physical changes noted in X. laevis larvae in the 

Langerveld et al. 2009 study and in their companion study (Zaya et al., 1011) lead to the 

hypothesis that tadpoles exposed to atrazine at 200 and 400 μg/L experience an 

imbalance in their energy homeostasis. They conclude that based on the Langerveld et 

al. 2009 study, a number of metabolic pathways were significantly changed in NF Stage 

62 larvae exposed to 400 μg/L throughout their development and that there was 

increased gene expression for pathways associated with conversion of lipids and proteins 

into energy (including synthesis and degradation of ketone bodies, the urea cycle, 

regulation of authophagy, citric acid cycle, glutathione metabolism and metabolism of a 

number of amino acids. Decreased gene expression was associated with carbohydrate 

metabolism, fat storage, protein synthesis, glycolysis/glucogenesis, fatty acid elongation 

in mitochondria and ribosomal proteins. The authors considered these changes reflective 

of the changes in body weight and fat body size noted in the Langerveld study and that 

some of these changes were replicated in the Zaya et al. companion article. The authors 

note that the atrazine-related increases in gene expression associated with ketone body 

synthesis/degradation is an adaptive response to starvation at NF Stage 62 and that it 

appeared that these animals depleted their glucose stores and shifted transcriptional 

responses to increase fatty acid oxidation to accommodate energy needs and that this was 

consistent with increased fatty acid β oxidation. The authors acknowledge that the 

increased expression of glutathione metabolism could be a result of the fact that excretion 

of atrazine is accomplished after Phase I metabolism followed by conjugation to 

glutathione and would be associated the utilization (mobilization) of amino acids. 

The absence of significant effects on ATP or ADP:ATP ratios was attributed to the 

successful effort by animals to respond adaptively to stress. The authors note that the 

increase in rate of apoptosis noted in the liver from NF Stage 62 larvae implied that 

energy stores were not detrimentally depleted since the process of apoptosis requires 

energy to occur. 

The authors conclude that based on changes in gene expression, larvae exposed to 

atrazine at 400 μg/L experience an energy imbalance starting early in exposure and 

continuing throughout exposure and that although the etiology of this energy imbalance is 

unknown, it could be due to detoxification and excretion of atrazine and/or endocrine 

disruption, the later through stimulating hormonally-mediated mobilization and increased 

β–oxidation of lipids. Changes in body weight early in exposure showed that atrazine 
245

significantly altered normal physiological functioning of the animals. Animals where 

exposure continued through NF Stage 62 were smaller and had decreases in fat body size. 

Description of Use in Document (QUAL, QUAN, INV):

Qualitative 

Rationale for Use: 

The etiology of the effects on gene expression reported in this article are uncertain; 

however, the study demonstrates up-regulation of certain genes and down-regulation of 

other genes. Given the difficulties with the Langerveld study though, the information 

obtained from the gene expression is of questionable utility; whereas, gene expression 

information from Zaya studies C and F also provide gene expression information. 

Although the concentrations tested, i.e., 400 μg/L, may not be environmentally relevant 

except in in-field puddles, it is possible that the observed effects may be associated with 

the energy demands of metabolizing atrazine. The companion article by Zaya failed to 

show any significant effects on length, but did demonstrate significant reductions in body 

weight, liver weight and fat body size. 

Limitations of Study: 

While atrazine residues of 200 - 400 μg/L may be detected in puddles and ephemeral 

pools located in treated fields, these do not reflect values typically reported in surface 

water adjacent to field treated with atrazine. As such, the "environmental relevancy" of 

these treatment levels is uncertain. 

The article refers to Langerveld et al. 2009 to support claims that atrazine exposure at 

400 μg/L resulted in significant reductions in survival and growth (weight, length and fat 

body size). However, the Langerveld et al. study has some major uncertainties associated 

with it especially given that so many of the test animals were not surviving to 

metamorphosis that the protocol had to be adjusted to work with younger animals. This 

further suggests that environmental conditions in the study may not have been sufficient 

to support animal survival. In studies where environmental conditions are poor and test 

animals are struggling to survive, it is uncertain whether any meaningful relationships 

can be evaluated for specific chemicals within that environment. 

Tadpoles were reportedly exposed from post-hatch Day 5 (NF Stage 47) presumably 

since this would correspond to when Xenopus laevis larvae (tadpoles) initiate feeding. 

Exposure proceeded to NF Stage 62 and would correspond to metamorphosis when it is 

likely that test animals would cease feeding and begin to resorb tissues to sustain the 

metamorphosis process. As such, catabolism of fats and proteins is likely. By NF Stage 

61 the filter apparatus of X. laevis larvae starts to degenerate and at Stage 62, the entire 

filter apparatus shows signs of cytolysis. 
246

The researchers apparently selected analysis samples based on the highest levels of 

expression. This constitutes judgment sampling and not random sampling. It is uncertain 

whether differences identified in this study would have been apparent had random 

sampling been used. The authors discuss a potential energy imbalance as a result of 

atrazine exposure; however, energy balances are difficult to assess. The authors’ 

conclusions that the etiology of the "apparent energy imbalance" could be due to 

detoxification and excretion of atrazine and/or endocrine disruption is broad, and with 

respect to endocrine disruption, it is speculative. Given that the authors acknowledge that 

the glutathion [detoxification] pathway on which atrazine is subject to is energy 

demanding, it may be a more plausible explanation for the effects they report. It is 

uncertain whether the study results support the statement that "clearly, acutely and 

chronically exposed tadpoles are compromised, decreasing their ability to survive the 

stresses of metamorphosis and/or to adequately support reproduction. The preceding 

work (Langerveld et al. 2009) cited by the authors suggests that the test animals may be 

more challenged by the study conditions than by atrazine. Citing "near-significant" 

results has little utility as there is no way to determine short of additional samples 

whether the effect is by chance alone. 

Although the authors state that Studies C and F resulted in depleted fat stores, the 

preceding publication by Zaya et al. 2011 did not indicate any significant difference in 

histological lipid staining; fat bodies in larvae exposed to atrazine at 400 μg/L were 

significantly [smaller]; however, it is unclear what that reduction in percent of body 

weight was associated with. Additionally, the authors authors state that the rate of 

apoptosis was increased in NF Stage 62 larvae; however, the companion article by Zaya 

et al. only speculated that increased caspase-3 activity would lead to increased apoptosis 

in spite of the fact that no pathology was observed through liver histology. 

 

Primary Reviewer: 

Thomas Steeger, Ph.D., Senior Science Advisor, Environmental Risk Branch 4, 

Environmental Fate and Effects Division, Office of Pesticide Programs 

Secondary Reviewer (required if study results are used quantitatively):

Not applicable 

 
247

4. References from the 2003 and 2007 SAPs on Amphibians 

2003 (From Table A in the 2003 While Paper) 

2007 SAP (next page) 
248