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 uncer tainties 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 Amphibian s 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 SUMMARI ES (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-C oll, 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., Scha effer, 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. Spr inger, H. Hrueger, J. Wolf, L. Holden, and A. Hosmer. 2009 .................................................................................1152 3.26. Kloas, W., I. Lutz, R. Urbatzka, T. Spr inger, 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. Mi yahara, 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. Sesterh enn, 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. Si h, 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. Whitak er, S. Kohler and C. F. Ide. 2011 ............... 236

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

4. References from the 2003 and 2007 SAPs on Amphibians ...................................... 2473 1. Table A. Summary of Available Amphibian s Open Literature Data (that passed the ECOTOX and OPP screen)4Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)EffectAllran & Karasov2000 / E56572Rana pipiens/Nasco(unknown if Lab/field /TP (G25) to M(138 d total)T /99NA /(Y/NA);NSNS20 TP/7L;48 hrMetamGroBio/MolSurv192 (M)192192192>192>192>192>192% & time to metamorphosisMass; SVLHematocrit% mortalityMetamorphosis and growth endpoints based onsubset (50%) of organisms and uncertainty inability to get a true randomization based oninformation presented; summary data for eachtreatment group for mortality and hematocrit notreported (only reported mean & SE for allreplicates combined); ammonia levels not reportedQualitative-Lower level ofconfidence for growthand metamorphosis endpoints based on subsampling as cannot discount potential confoundingfactors and also for mortality and hematocrit as notreatment summary information presentAllran & Karasov2001 / E59251R. pipiens & R.sylvatica &Bufoamericanus /Field /A(R.pipiens)for 14 dand E for 96 hrphT /99NA /(Y/NA);NSGlass (E) /NS (A)50 or 30E/0.075L(E); 24 hrMetaGroBehav (E)Behav (A)Gro (A)Bio/Mol(A)Surv2590200002000025902000020000200004320>20000>200004320>20000>20000>20000Deformities (tail, facial edema)(E)Mass (E)Swimming (E)Buccal ventilationMass (A)HemoglobinHatch and mortalityAtrazine measured at organism collection site;water quality parameters not specified-authorsstate conducted according to ASTMQualitative-Lower level ofconfidence as lowlevels of atrazine measured at organism collectionsiteBirgeet al. 1980 /E6187R. catesbeiana,R. pipiens, R.palustris, B.F /80NS / (Yunsure ifNC orglass50 -103 E/ 0.5L; 2.5hSurv 4dph LC50-- --R. catesbeiana= 410R. pipiens= 7680R. palustris= 17960LC50s were not based onmortality per se, but onabnormalities that would reportedly precludesurvival under natural conditions; solvent (if used)1TP = tadpole; LV = larvae; M= metamorphorsis; E= embryo; G = gosner stage; NF=Nieuwkoop and Faber;SVL=snout-vent-length; A = adults; H = hatch; J =juvenile; if reported in study, developmental stage or some other identifier at test initiation is given, if not reported thandesignated as NS(not specified);2TS = Test substance; T = technical; F = formulation3NA = not applicable; NS = not specified; ACTN = acetone; EtOH=ethanol; DMSO=dimethylsulfoxide; MeOH=methanol; IPA = isopropylalcohol4Negative Control (NC) and/or Solvent Control (SC) included in study design; MA in CNT = Measurable atrazine in control treatment group(s)- Yes (Y) or No(N) or Not Specified (NS)5The values reported represent loading reported as mass (g) or number of larvae/tadpoles in a volume of water (L). If loadingwas reported as number oflarvae/tadpoles in a certain test vessel size but test solution volume was not reported, then this is presented # organisms intank (volume reported if available) asactual loading could not be determined; PE = polyethylene6Water7Metam = metamorphosis; Gro = growth; Imm/Inf = immune system or infection; Bio/Mol = biochemical/Molecular; Sexdev = sexual development;Behav=behavioral; Surv = survival; N = nominal conc.; M = measured conc; NDR = not dose responsive (no significance at higher dose).5Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effectamericanus/NS (purchased)/ E for 4 dphSC); NSB. americanus>48000 was not identified; formulation used so uncertaintyin source of toxicityQualitative – Higher levelof confidence; howeverLC50 values are combination of both lethality andabnormal effects and formulation usedBrodeuret al. 2009/ E112588R. arernarum/LP8/ TP(G38/39)-MT/98NA /(Y/NA9);Nglass10 TP/0.04L; 48hrMetamSurv(LC50)NDR (N)NA100NA↓&↑Time metamorphic stage and/orduration between stagesLC50 (stage 4) = 7150-25370LC50 (stage 25) = 2320-27160LC50 (stage 38-39) = 12630-18270(LC50s range from 4 d to 28 d)No water quality parameters reported;metamorphosis results not dose responsive (notsign. at 1000 or 5000 μg/L depending on metamendpoint (100 lowest conc tested); hypothesistesting for effects on metamorphosis conductedusing EC50 valuesQualitative-Lower level of confidence based onhigher loading with no water quality parametersreportedBrodkinet al2007 /E103351R. pipiens/Field (ANA10) /A for 8 dT/NSNA / (N &N11/NA);NSplastic NA; 24 hrImm/infSurv<0.01(N)100.01>10↓White Blood CellsStudy indicated atrazine did notcause mortality; not explicitlyreported in terms of NOEC/LOECAdult frogs cultured using Styrofoam rafts;uncertainty in how organisms were given whattreatments; effects are not clearly andquantitatively linked to the assessment endpointsof growth, reproduction, and survivalInvalid based on lack dilution water only control(dilution water but subject to immune treatmentsused)Chounget al.2011a / E153858Litoriaraniformis/Lab / TP (G26)for 10 wksT/>98NA /(Y/NA);NSglass10 TP/10L;2X/wkMetamGroSurv25 (N)25-->25>25--Time to metamorphosisMass; SVLStudy indicated survival >97.5% forstudy; not explicitly reported interms of NOEC/LOECLimited reporting of potential contaminants infood and water; number of egg masses used notspecified; measured ammonium (not NH3)Qualitative-Higher level ofconfidence; howeverthere is a lack of reported measurements in foodwater and number of egg massesClementset al.1997 / E20274R. catesbeiana /CBSC / TP for24 hrF /88.2NA /(Y/NA);NSglass NS; NABio/MolSurv<4800(4200 ai)--4800(4200ai)↑DNA damage in erythrocyctesNS; reported testing the maximumsurvival concentration and dilutionsLimited reporting on water quality parameters andtest design (# of replicates); conducted abovewater solubility8LP = adults used to collect eggswere obtained from local provider9Test concentrations 1-25 mg/L preparedwithout solvent; 30 mg/L prepared using acetone (0.2%) sosolvent control (0.2% acetone) was also included in design10ANA=Amphibians of North America (Nashville, TN, USA)11A treatment group that was only exposed to dilution water (andnot injected with Ringer solution or subjected to thioglycollate stimulation) was not included.6Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect--from that conc.Qualitative – Lower level of confidence based onlack of test design parameters and water qualityreportedCoadyet al2004 /46965605R. clamitans/Field / TP(G25)-G42 (273d)T/97.1EtOH(0.005) /(Y/Y); YNS30 TP/4Lthen 16L;72 hrMetamGroSurvNDR(M)282811>28>28↑Time to metamorphosis at 11 butnot 28Mass; SVL% mortalityPoor water quality; high control mortality; lack ofresponsiveness to positive control (estradiol)Invalid based on control contamination; highcontrol mortalityCoadyet al2005 /46965602Xenopuslaevis/Lab/ TP(72 h ph)- MT/97.1NA /(Y/NA);Yglass30 TP/4L;50% every72 hrGroSexdevBio/MolSurv25250.125>25>251>25Mass; SVLGonad development; laryngealmuscle↓estradiol males% mortalityHigher loading with 50% renewal of solution at72 hr may have promoted poor water quality;delayed metamorphosis in controls; statisticaldifference between controls (possible confoundingfactor); lack of responsiveness to positive control(estradiol)Invalid based on control contaminationForson & Storfer2006a / E82033A.macrodactylum/ Field / LV (ca.6 wks) for 30 dT /86.5NA /(Y/NA);NSPE1 LV/0.5L;3X/wkMetamGroSurv18.4 (M)18.4184184184>184↑larval period↓mass & SVL% mortalityLimited reported water quality;Qualitative-Lower level ofconfidence due to lackof reported measurements incontrol with slightlyhigher than recommended loading with limitedwater quality and a renewal rate of 72hrperiodicallyForson & Storfer2006b / E103072A. tigrinum/Lab / LV(12wks)-M(111 d total)T /NSNA /(Y/NA);NSPE1 LV/0.5L;every 3daysMetamGroImm/infSurv160 (N)1601.6(NDR);16-->160>16016;160--Time to metamorphosisMass, SVL↑infection rate;↓leukocytesReported 31 mortalities (384exposed, 8% mortality); results notpresented in terms of NOEC/LOECLimited reported water quality; infection rate notdose responsive (not sign at 160 μg/L) ; potentialstress from repetitive mass measurementsQualitative-Lower level ofconfidence due toslightly higher than recommended loading withlimited water quality anda renewal rate of 72hrFortet al. 2004 /E81751X. tropicalis &X. laevis /Xenopus I(adults) / E –NF46T/NSDMSO(1%) /(Y/NS);NSNS20E/0.01L;24 hrMetamSurv(FETAX)-- --X. laevisLC50 = 24500 or 25600EC50 = 4100 or 4400X. tropicalisLC50 = 19400 – 31800EC50 = 2100 - 9300Limited water quality reported; use of notrecommended solvent and high conc (althoughallowed under ASTM FETAX assay guideline)Invalid based on solvent choice and concentrationFreeman &Rayburn 2004 /B. americanus& X. laevis/T /NSNS / (Cused NS ifNSNS (2TP/3LBio/MolSurv800NS>800NSNuclei (represent genotox)Not specified; based on reported ‘N’Stats for nuclei conducted on CVs; uncertainty insample sizes; no reported water quality; loading7Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)EffectE78644 Field(parents)(Bufo)& Nasco / TP(NF NS) for 3-5wksNC, SC);NStank);weeklyin tables appears 100% survivalunknown (fill volume not reported)Qualitative-Lower levelof confidence due touncertainty in sample sizes and loading unknownwith no reported water quality and relatively longrenewal periodFreeman &Rayburn 2005 /E81459X. laevis/Nasco/XenopusExpress / TP(varied NF40 to54) for 3-5 wksT /98NA /(Y/NA);NS12NSNS (1TP/3Ltank);weeklyMetamGroBio/MolSurv<100 (N)800Varied--100>800800--↓metamorphic stageMass↑flow cytometric analysis meant toillustrate metemorphosis anddevelopment); results varied basedon test% Mortality (0-16.7%) for differentassays, reported as occurringrandomly in treatments; mortality interms of NOEC/LOEC not reportedStats for flow cytometric conducted on CVs;uncertainty in weight analysis with regards toaccounting for different stages of organisms; fillvolume of test vessel (3L) not reported howeverwater quality appearsto be acceptableQualitative- Medium level of confidence formolecular endpoint due to analysis using CV andother reasons described for metamorphosis;Invalid for weight due to potential confoundingfactor; Qualitative –Higher –for metamorphosisstage; however, lack of analysis of atrazine incontrolFreemanet al. 2005/ E81460Bufoamericanus/Field (Adults) /TP (G20 or 25)for 3 or 4 wksT/98NA/(Y/NA);NSNSNS (2TP/3Ltank);weeklyMetamGroBio/MolSurvNDR (N)NDR10000--250800>10000--↑Stage at test completion↓Mass at test terminationMean total # of nucleiReports no acute toxicity observed;mortality in terms of NOEC/LOECnot reportedWater quality parameters in study not reported; fillvolume of test vessel (3L) not reported-unable toverify loading; metamorphosis and mass not doseresponsive (metam-not sign at 800 μg/L in othertest different age at initiation; mass- not sign at800 in other test or 250-10000 in another test)Qualitative- Lower level of confidence forMetamorphosis and Bio/Mol- due to loading notdefined and no water quality parameters withweekly renewal; Invalid for weight due topotential confounding factorGibble & Baer2011 / E153856X. laevis/Nasco / TP(NF49/50)-M(90 d total)T/98 orF/40.8DMSO(0.0173) /(Y/Y); N(Tech),NS(Form)NS1g/L [perday –reviewerassume]for T / NSfor F;7X/day orMetamGroSurvImm------986 (N)------>986Specific parameters within theseendpoints not reported formetamorphosis, growth, survivalPeptide activity (expressed as totalprotein)Atrazine measured in solvent control (exposed forpotentially 48 hours) in atrazine technical study;survival not reported; study design (replicates, #organisms) not reported for the formulation;appears frogs used as experimental unit, not tanks,for peptide assay for tech atrazine (unknown forformulation)12Study reports that solutions measured from experimental tanks and atrazine concentrations werewithin expected range; howeverunsure if this included thecontrol8Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect3X/wkInvalid for growth, survival and metamorphosisbased on lack of reporting results and controlcontamination; Qualitative-Low level ofconfidence- for peptide bioactivity analyses foratrazine formulation based on lack of survivalreporting and test designGoleman & Carr2003 / 45867707X. laevis/XenopusExpress(Adults) / TP(48 hph)-NF66(78 d total)T/98.6ethanol(0.0025)-E2 grouponly /(Y/Y);NglassVaried 60-65 TP/0.250 –4L; 50%every 72hrMetamGroBehavSexdevSurv19.5 (M)19.510.310.319.5>19.5>19.519.519.5>19.5Forelimb emergence, tail resorptionMass; SVL↑abnormal swimming↑ovotestes% mortalityWater quality concerns; lack of response topositive control and potential delayeddevelopmentInvalid based on appeared lack of response topositive control and potential overall delayeddevelopment in the studyGundersonet al2011 / E153613R.catesbeiana/Field / TP (NS)for 2 dT/NSIPA(0.001) /(N/Y); NSglass0.63 g/L;static(48hrexposure)Bio/MolSurv10-->10--Relative fold difference for CYP19and SF-1Reported no mortalities in study notreported in terms of LOEC/NOECLimited water quality reported; while no sign diffin CYP19 and SF-1 for each treatment, correlationof CYP19 and SF-1 m RNA in the brain whencombining 1 & 10 ppb resulted in sign diff.Invalid based on use on solvent choice (notrecommended solvent) and no negative controlused in studyHaridonet al. 1993/ E4324Pleurodeleswaltl /LV for12 dT /NSDMSO(0.05) /(NS/NS);NSglass15 LV /1.5L; 24hrBio/MolSurv300NS>300NSMicronuceated RBCs (genotox)Reported that maximumconcentration was dose not to inducedetectable physiological disturbancesfor 12 days of treatmentLimited water quality reported; Used DMSO athigher than recommended conc.; uncertain in whatthe control was comprisedof; uncertain if usedpsueoreplication in resultsQualitative – Lower levelof confidence due tosolvent choice and concentration and higher thanrecommended loading with limited water quality,also uncertainty in control and statistical analysisadd to uncertaintyHayes 2002 /E70207X.laevis / Lab /TP(NF46)-M(NF66); A for46 dT/NSEtOH(0.004)(N/Y);NS13plastic30 TP /4L;72 hrMetamGroSexdelBio/MolSurv200 (N)2000.01;0.8<25200>200>2000.1;125>200Time to metamorphosisMass; SVL↑Ovotestes;↓Laryngeal dilator↓Plasm testosteroneMortalityLimited water quality reporting; use of plastic testvessels; indirect measured of effect (laryngeal); inreport results on metamorphosis, growth andsurvival not reported in studyQualitative – Lower level of confidence based onhigher loading with limited water quality reportingand a renewal period of every 3 days13Study indicates concentrations confirmed by two laboratories; however, unclear if this included the controls.9Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)EffectHayeset al. 2003R. pipiens /Field / (48hrpost hatch)-MT /98EtOH(0.0036) /(N/Y)NS14NS30 TP /4L; 72 hrMetamGrowthSurvSexdevNSNSNS<0.1NSNSNS0.1Results not presented in report formetamorphosis, growth, survival↑male gonadal dysgenesis(underdeveloped); Sex reversalNo statistical results associated with sexualdevelopment endpoints ; study reported atmetamorphosis growth measurements taken butresults not presented in studyQualitative – Lower level of confidence based onhigher loading with limited water quality reportingand a renewal period of every 3 daysHayeset al2006aX.laevis / Lab /TP (4d posthatch)-M(NF66)T/NSMeOH(0.004) /(Y/Y); NSplastic30 TP/4L;72 hrSexdev Uncertainty if animals exposedto atrazine in this study orif from another study. Mayhave used different clutchesto make comparisonsSex ratio of controls skewed; lack of a consistentand clear dose response; limited water qualityparameters reported.Invalid due to uncertainty if animals were exposedto atrazine in this study or were from another studyHayeset al. 2006b /E85815R..pipiens /Field (Adult)/TP (48hr posthatch)-G46; X.laevis /Lab /Afor 27 dT/98EtOH(0.0036) /(N/Y); Nplastic30 TP /4L;72 hrMetamGroImm/infSurv0.19 (M)<0.19<0.190.19>0.190.190.19>0.19Time to metamorphosis↓Mass; SVL↑Thymic plaques (X. laevis)MortalityWater quality parameters not reported; reportedresults are inconsistent with the data presented inreport; authors rely on the lack of statisticalsignificance to draw conclusions.Qualitative –Lower levelof confidence based onhigher loading with limited water quality reportingand a renewal period of every 3 days as well asinconsistent reporting adding to level ofuncertaintyHayeset al2010 /E153853X.laevis/ Lab /H-A (up to 3yrs post M)T/NSEtOH(0.003) /(N/Y); NSplastic30 TP/4L;72 hrSexdevBio/MolBehavSurvMetamGro<2.5(N)<2.5<2.5NSNSNS2.52.52.5NSNSNS↓Breeding gland, laryngealdevelopment↓Testosterone↓Mating behaviorResults on survival, metamorphosisand growth of animal not reportedIn general, the study does not describe and/oraccount for all of the animals used in eachtreatment/replicate; uncertainty in reported results(e.g., # of phenotypic female/genotypic malecompared to total exposed); Results for survival,metamorphosis and growth not reportedQualitative – Lower level of confidence based onhigher loading with limited water quality reportingand a renewal period of every 3 days anduncertainty in reported results add to level ofuncertainty; Invalid forsurvival, growth andmetamorphosisHeckeret al2005a/ E79287X.laevis /XenopusExpress / A forT/97.1NA(Y/NA);NNS1frog/10L;3 daysSexdevBio/MolSurv258(M)24.6-->258258--Gondal abnormalities↓Plasma testosterone4 total mortalities (65 exposed)Uncertainty in methods for weights (and itcascading effect) and gonad evaluation; LOD/LOQnot reported for steroid analyses; unclear about14Study indicates concentrations verified; however, unclear if this included controls10Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect36 dreported; mortality nor presented interms of NOAEC/LOAECsample size for steroid analysesQualitative-Lower levelof confidence due touncertainty in methodology for weights, gonadevaluation and biochemical analysesHeckeret al2005b/ E79288X.laevis /XenopusExpress / A for49 dT/97.1NA(Y/NA);Nfiberglass4.9 g/L;3daysSexdevBio/MolSurv<11.6 M107--11.6>107--GSIPlasma testosterone; estradiol1 total mortality reported; mortalitynot presented in terms ofNOAEC/LOAECNo solvent control for positive control; Somewater quality reported but no reporting of nitrates,nitrites, and ammonia; limited sensitivity foraromatase assayQualitative – Lower level of confidence based onhigher loading with limited water quality reportingand a renewal period of every 3 daysHoweet al1998 /MRID 45202910R. pipiens, B.americanus /F /TP (G29 & 40)for 96 hF /40.8NA(Y/NA);Nglass20 TP /15LSurv 96 hrLC50-- --LC50 valuesR. pipiens(early stage) = 47600R. pipiens(late stage) = 14500B. americanus(early) = 26500B. americanus(late) = 10700Control survival not reported; no raw data; testedabove water solubilityQualitative – Medium level of confidence based onlack of control data and only LC50 values reportedand testing above water solubilityHouck & Sessions2006 / E117318R. pipiens/ NS/ A for 4 or 8wksNSNS /(NS/NS);NSglassNA; 3daysImm/infImm/infSurv<1(N)10-->10>10--↓plaques representing antibodysecreting cells (4 wks)Number of Lymphocytes1 mortality (believed 24 exposed)reported; mortality not presented interms of NOAEC/LOAECNo information provided on culturing conditionsand limited information on experimentalparameters; uncertainty inwhether used a solventor not as test solution preparation not reported;uncertainty in source of frogs; frogs were reportedto not be feeding in trial 2; analyticalmeasurements of treatment groups not reported;uncertainty in how animalsin Trial 1 were used instudyInvalid based on frogs not eating in trial 2 andoverall lack of culturing and experimentalparameters and uncertainty in reported results inTrial 1Kerby and Storfer2009a / E117314A. tigrinum/Lab / LV(4wks) for 14 dT/NSMeOH(0.02);(N/Y); NSplastic1 LV/0.5L;1X/weekImm/infSurv200 (N)200>200>200Infection rate% Survival basedon atrazine alone,reported interactive effect (p=0.03)atrazine-virus with atrazine slightlyincreasing survival w/o virus andslightly decrease survival w/virusUncertainty in affect ofdead larvae on measuredviral load that was usedin analyses; limited waterquality reportedQualitative-Lower level of confidence givenhigher than recommended loading rate withlimited water qualityreported and a weeklyrenewal periodKiesecker 2002 /E68326R. sylvatica/Field /TPT /NSACNT &DMSOPE1 TP/3.5L;48 hrImm/infSurv<3NS3NS↓eosinophils↑trematode infectionsMortality not reported; based onUncertain if cercariae usedfor infection trial wereexposed to atrazine, therefore there is some11Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect(G25) for 4 wk(NS);(Y,Y); NSsample size in Figure 2 in reportsurvival appears to be 100%uncertainty in the environmental relevance of thisscenario; dilution water only group (no exposureto cercariae not used; limited water qualityreported; uncertainty in statistical analysisInvalid based on lack of dilution water onlycontrol, also due to environmental relevance withdifferent exposure scenarios between tadpole andparasite, unsure of statistical analyses adds touncertaintyKloas 2009a /E112914X. laevis/Xenopus I / TP(8 dpf) for 83 dT /NSNA /(Y/NA);Y15glass<1g/L/day;7X/dayMetamGroSexdevSurv100 (N)100100100>100>100>100>100Time to metamorphosisMass; SVL>10016Sex ratio; gonadal development% SurvivalAtrazine contamination of controls; markedfluctuations in water quality parameters;histological endpointcomparisons not maderelative to amphibian referenceQuantitative based on fact that althoughdeficiencies, study design and reporting wererobust enough to properly evaluate resultsKloas 2009b /153612X. laevis /Lab /TP (NF 48 and58) for 4 dT /NSNS /(NS,NS);NSglassNS (25TP/ 10Ltank); 24hrBio/MolSurv25NS>25NSAromatase, LH, FSH, 5-alphareductaseMortality not reportedAnalysis based on n=8, however only twoindependent tanks tested(removed 4 from eachtank that contained 25 tadpoles at initiation);limited water quality parameters reported duringtest, test solution preparation and what constitutesthe control was not reportedQualitative – Low level of confidence based onlack test design reportingKoprivnikaret al2007 / E103348R. sylvatica/Field / TP(G28/29) for ca.31 dF /40.8NA /(N17/NA);NSplastic1 TP/1.8L; 72hrsImm/infSurv3 (N)3030>30↑intensity of infection (frogsexposed but cercariae not)MortalityControl survival was 61.9%; Dilution water onlytreatment (no cercariae) was not used; replicatevariability not reported;effects only observedwhen tadpoles, not cercariae were exposed toatrazine –uncertainty in environmental relevance;limited water quality reportedInvalid based on lack of dilution water onlycontrol, lack of reported replicate variability andadverse effects observed only when tadpoles, not15Atrazine detected in one out of 4 blocks of negative control tanks in one of the studies; it was excluded from analysis16Significant 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 ofthe two reported studies; Noeffect on wt and SVL reported in other study; EFED does not consider the differences in wt and SVL to be a concentration-dependent effect17A dilution water only control was not included. A control that contained no atrazine but cercariae that were also not exposedto atrazine was used in the study12Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effectcercariae, were exposed (unknown environmentalrelevance) adds to the level of uncertaintyKoprivnikaret al2010 / E153870R. pipiens/ Efrom CBSC18/TP (<G27)- ~G4319T/99.2EtOH(100ug/L)/ (Y/Y);NSplastic1 TP/L;weeklyMetamGroSurv<3 (N)<3<3333Developmental stageMassMortalityVariability in mortality across 3 ppb treatmentgroups; uncertain if developmental stage wasaccounted for in mass endpoint; uncertainty inreported results in figures; uncertainty iforganisms were field collected or laboratory rearedInvalid based on uncertainty in reported resultsbased on Figures in report and limited reportedwater quality with relatively long renewal periodand variability in mortality across treatmentgroups add to uncertaintyLa Fiandraet al.2008 / E103336H. versicolor/Lab / TP (G25)-30 d past MF/88.2NA;(Y/NA);NSglass3 TP/7L;50%1X/wkMetamGroSexdevSurv195 (M)195195195>195>195>195>195Larval periodMass; SUL20Sex ratio; gonad development% SurvivalLimited water quality reported; uncertainty ifadults used to obtain eggs for culturing were fromthe field or laboratoryQualitative-Lower level ofconfidence, whileloading was within recommended limit, limitedreported water quality with relatively long renewalperiod(50% renewal every week) may havepotential environmental condition issuesLangerveld 2009 /E121497X. laevis/ Lab /TP (NF43) toNF46T/99.4NA /(Y/NA);NSPyrex4 TP/1L;every 2-3daysGroBio/MolSurv<400 (N)<400<400400400400↓Mass; SVLExpression of multiple genes% SurvivalLimited water quality reported; Uncertaintyregarding overall mortality in control and 400 ppbhowever, substantial mortality observed at 400ppb when approaching stage 63, therefore maynot be unexpected to observe significant sublethalchangesInvalid for growth and bio/mol due to significantmortality compared to control in the only atrazineconcentration tested alsohigh loading rate withlimited water quality and renewal rate periodically>48 hours; Qualitative Lowlevel of confidence forsurvival based on limited water quality, loadingand renewal rate.Larsonet al. 1998 /E60632A. tigrinum/Field / LVT /NSACTN(NS)/PE1 TP/4L;50% atMetamGro<81.8(M81.881.8>81.8↓metamorphosisAtrazine measured in controls; lower dissolvedoxygen reported18CBSC = Carolina Biological Supply Company (Burlington, NC)19G43- first observance of reabsorbed tail20SUL = snout-urostyle length13Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect(SVL= 30mm)for 86 d(N/Y); Y 48hr Bio/MolSurv<81.8--81.8--Mass; SVL↑plasma thyroxine;↓corticosteroneReported no exposure relatedmortalities; mortality not reported interms of NOAEC/LOAECInvalid due to control contamination and solventconcentration not reported and no negative controltestedLenkowski &McLaughlin 2010 /E153851X. laevis/ Lab /TP (NF41) for6-48 hrT/NSDMSO(0.1-0.35)/ (Y/Y);NSNS13-18 TP/0.01L; 24hr (48 hrexposure)Bio/MolSurv350(N)NS3500NS↑MMP18 expressionLimited reporting on test elements (replicates,solution preparation, testvessels, analysis of testsolutions, water quality parameters, loading,control survival)Invalid due to solvent concentration and solventchoice and lack of reporting on test design andexperimental parameters contributing to higherlevel of uncertaintyLenkowski 2010 /E153876X. laevis/ Lab /TP (NF41) for48 hrT/NSDMSO(0.1-0.35)/ (Y/Y);NSNS13-18 TP/0.010L;24 hr (48-hrexposure)MetamSurv350 (N)--3500--↑Malformations in intestines andcranial cartilageReported no mortality in atrazineexposures, control mortality notexplicitly reportedLimited reporting on test elements (solutionpreparation, test vessels, analysis of test solutions,water quality parameters)Invalid due to solvent concentration and choiceand lack of reporting on test design andexperimental parameterscontributing to higherlevel of uncertaintyLenkowskiet al2008 / E103268X. laevis/ Lab/TP (NF41-46)for 48 hrT /98DMSO(0.1-0.35)/ (Y/Y);NSNS15 TP/0.01L; 24hr (48 hrexposure)MetamSurv<10,000(N)--10000--↑visceral bleeding & organmalformationsMortality reported as <5%Limited reporting on test elements (test vessels,analysis of test solutions, water qualityparameters)Invalid due to solvent concentration and lack ofreporting on test design and experimentalparameters contributing to higher level ofuncertaintyOka 2008 /E103345X. laevis/ Lab /TP (NF49)-M(56 d total)T/97.4EtOH(0.01) /(N/Y); NSglass30 TP/10L;3X/wkMetamBio/MolSexdevSurv100 (N)1001-->100>10010--Time to metamorphosisAromatase↑female ratioReported≥survival in controls andsome treatment groups with lowsurvival in one of two tanks for twoconcentrationsEstrogenic activity measured in parental food;variable replicate mortality which resulted in onlyone replicate for analysis for two treatment groupsfor sex ratio endpoint; no statistical resultsreported for metamorphosis; limited water qualityreportedQualitative- Lower levelof confidence based onhigher loading with limited water quality reportingand an occasional renewal period of every 3 dayscoupled with variable mortality in tanks for twotreatment groups (not dose responsive); Invalid formetamorphosis as statistical results not reported.14Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)EffectOlivier & Moon2010 / E153629A.maculatum/Field / E for ~76 dF/42.6NA /(Y/NA);NSplastic1 eggmass/0.750L; 3wksMetamSurv50 (N)50100100Developmental stage before death orhatch% Hatch; mortality reported as eggmass mortality specific stats notreportedTemperature regulator malfunction in whichpathologies were observed in treatment groups(not control) shortly thereafter and mortality inatrazine treatments occurred shortly after solutionrenewal; Uncertainty in number of embryos pertreatment and evaluation of oxygen depletion;limited water qualityQualitative-Lower levelof confidence as itappears that additional potential stressors oftemperature change and solution renewal mayhave contributed to observed pathologies as wellas limited water quality reported with longrenewal period (21 d)Ortonet al2006 /E82034R.pipiens /CarolinaBiological /Taylor-KollrosStage 2/3 –23.24T/98.2NA /(Y/NA);NNS20 TP /8L;75% at 48hrMetamGroSexdevSurv15 (M)15<15<15>15>151515Time for forlimb emergenceSVLSex ratio; gonad develop% mortality; lower in treatmentgroupsHigh control mortality (ca. 50%); higher incidenceof intersex (12.5%) in controls; sex ratio ofcontrols appears skewedInvalid based on higher incidence of reportedgonadal intersex in control and higher controlmortalityMorgan 1996 /E63246X. laevis/Xenopus I(adults) / E for96 hrF /40.8NA /(Y/NA);NS21plasticNS (20E/petridish) ; 24hrMetamSurv-- 11000or 1100(4488or 448ai)LC50 = 100000 or 126000 (40800or51408 ai)EC50 (abnormalities) = 33000 or<8000 ( 13464 or 3264 ai)Limited water quality reported, test solution fillvolume not reported; conducted above watersolubility; formulation usedso source of toxicityunknownQualitative – Medium level of confidence basedon limited water quality reportedRohret al., 2003/E71723A. barbouri/Field / E for 37d (53 d totaltest)T /80DMSO(0.03) /(Y/Y); NSGlass10 LV/2L;50% at 48hrGroBehavSurv400 (N)40400>400400>400Mass; SVL; limb deformaties↑Locomotor activityTotal SurvivalSolvent control contained both acetone andDMSO whereas atrazine concentrations onlycontained DMSO (which is not a recommendedsolvent); author states organisms likely exposed toatrazine prior to test dueto agricultural activitiesaround collection site; limited water qualityreported; uncertain if potentially different stages attest termination (stagesnot reported) influencedgrowth analysisQualtiative- Lower level of confidence based onhigher than recommended solvent concentration21Test concentrations measured but do not report results for control; unsure if analyzed15Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effectusing a non-recommended solvent in addition thelimited reported water quality with the higherloading rate add to the level of uncertaintyRohret al., 2004 /E81748A. barbouri/Field / E-M(mean 64 d)T /80DMSO(0.03) &ACTN(0.03)22/(N/Y); NNS31 or 40E/9.5L;weeklyMetamGroBehavSurv4(N)40404040400400400↑time to metamorph↓mass↑locomotion activity;↓refuge use↓SurvivalWhite film on dead embryo unknown if funguscaused or followed mortality; larval survivaldifferences between years; although pH, temp, andDO ok, nitrates and ammonia not reported;Qualitative-Lower level of confidence based onhigher than recommended solvent concentrationusing a non-recommended solvent in addition touncertainty in the source ofmortality in the deadembryosRohr and Palmer,2005 / E81664A. barbouri/Field /Metamorphsfor 5 hrsT /80DMSO(NS) /(N/Y); NSNSNA; 5 hrexposureBehavSurv4(N)--40--↓water conserving behavior;↑activityReported no mortality; results notpresented in NOAEC/LOAEC termsThis study used metamorphs from previous study(Rohret al2004, E81748) in which organismswere exposed to different atrazine concentrationsand test subjects in this study were not exposed toany additional atrazine during the behavioral study(water conserving behaviors). See Rohret al2004(E81748) for commentsQualitative-Lower level of confidence based onidentified uncertainties in the other study fromwhich the organisms in this study were obtainedRohr 2008b /E112912R. clamitans/Field / TP(G24-26) for 14dT/>98ACTN(NS) /(Y/Y); NSglass4 TP/0.3L; NSImm/infSurv<201(M)201201>201↑% of encyted cercariaeMortalityTadpoles exposed to atrazine before and aftercercariae exposure but cercariae not-uncertainty inenvironmental relevance; for cercariae-tadpoleresults, the treatments arecompared to the solventcontrol suggesting a significant difference betweenthe controls; limited water quality reported;control mortality not clearly indicatedQualtiative- Lower level of confidence as controlmortality not clearly reported limited water qualityreported with a non-specified renewal period(tadpoles exposed for 7 days at a time) and higherthan recommended loading and environmentalrelevancy that adds to the uncertaintyRohret al. 2009 /E117315B. americanus/Field / LVT/99ACTN(0.0002) /NS6 TP/0.5L(96hr)BehavSurv196 (M)-->196--Fear queues (predator and parasite);activityNo negative control (authors report no effect fromsolvent based on previousdata); limited water22Solvent concentration was calculated by reviewer16Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect(G25-27) for 4d(N/Y); NS then1/3.5L;staticReported no effect on mortality withnearly 100% survivalquality reportedQualitative-Higher level of confidence, howeverno negative control andlimited water qualityRohret al., 2006 /E82038A. barbouri/Field / AT /80DMSO(0.03) &ACTN(0.03)23/(N/Y); NNS31 or 40E/9.5L;NASurv <4 4↓Long term survival (carryovereffects)This study used metamorphs from previous study(Rohret al2004, E81748) in which organismswere exposed to different atrazine concentrationsand test subjects in this study were not exposed toany additional atrazine to observe survival due tocarryover effects. See Rohret al2004 (E81748)for commentsQualitative-Lower level of confidence based onidentified uncertainties in the other study fromwhich the organisms in this study were obtainedRohret al. 2011 /E153803A.barbouri/Field / E-H(49d) or LV(NS)-M or 19 dT/99ACTN(8.25) /(N/Y); NSglass12 E/2L;weeklyGro(E)Metam(L)Gro (L)Surv40 (N)NSNS<4400NSNS4MassAuthors indicate effect on metamand growth but conc. not reportedEmbryos –↑Mortality; larvalexperiment- no significant foratrazine treatment groupLarval survival lower across all treatments andcontrol for study at 19°C; larval mass fromembryo study conducted using preservedorganisms; limitedwater quality reportedInvalid based on higher than recommendedsolvent concentration using a non-recommendedsolvent in addition to notusing a negative controlgroup; also limited water quality reporting withhigher than recommended loading and weeklyrenewal period add to the level of uncertaintySpolyarichet al.2010 / E153638L.tasmaniensis/ Field / TP(G28) to G42(4wks)T/98NA /(Y/NA);Nglass10TP/10L;2X/wkMetamGroSexdevSurv30 (N)3030-->30>30>30--Rate of developmentLengthSex ratio; gonad developmentReported 2.3% mortality in testoverall; mortality not reported interms of NOAEC/LOAECSex determination based on gross morphology;histology on male gonads only; Metamorphosisand length data analysis appear to be based usingthe individual frog as the experimental unit andnot the tank as data in figures appears to be onpooled results of the three trialsQualitative-Higher level ofconfidence as loadingseemed within recommended levels, however,ammonia and nitrate concentrations not reportedwith a 2X/wk renewal period and field collectedorganisms (Sydney Australia suburb) so priorexposure history is unknownStorrs andPseudacrisF / NA / PE 1 TP/ 0.1Surv NDR 2.84 Survival- not dose responsive, for all Survival not dose- responsive; animals that23Solvent concentration was calculated by reviewer17Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)EffectKiesecker 2004 /E78290crucifer, R.sylvatica,B.americanus,R.clamitans /Field / TP(early stageG25-27) & (latestage G29-36)for ca. 30 d85 (Y/NA);NSor 0.5L;every 3daysbut wood frog, report sign. effects at2.84, but not always at 25 or 64metamorphosed during the experiment wereeliminated from the data set; limited water qualityreportedQualitative-Lower levelof confidence due tosurvival response not dose-responsive and limitedwater quality reported withloading and renewalrate.Storrs & Semlitsch2008 / E103378H. versicolor;R.sphenocphala;B. americanus/Field / TP(G25)-MetamorphsT/99.2EtOH(0.0125) /(Y/Y); NGlass &plastic1 TP/0.8L; 72hrMetam &Sexdev(H. vers.)Metam &Sexdev(R. sphen)Metam &Sexdev(B. amer)Surv (all)25.1 (M)30.4125NS>25.1>30.4>125NSTime to metamorphosis; OvarianstageTime to metamorphosis; OvarianstageTime to metamorphosis; OvarianstageMortality not reportedLimited water quality reported; very slightestrogenic activity measured in dilution water;Uncertainty in number of animals exposedQualitative-Lower level of confidence based onlimited water quality reported with slightly higherthan recommended loading and renewal period of72hr as well as slightly higher than recommendedsolvent concentrationStorrs Mendezetal. 2009/ E118898B. americanus/Field /Metamorphs for2 dT/99.2ACTN(NS)/(NS/NS);NSNS NA; NABehavSurv1430μg/kg(N)NS>1430NSSoil choice preference (ATZ or noATZ)Limited reporting on control group preparationuncertainty regarding whether control containedsolvent; uncertainty in measured conc. due to lessthan nominal and long storage timeQualitative-Medium level of confidence based onlimited reporting for control preparationStorrs-Mendez &Semlitsch 2009/E153841H. versicolor;R.sphenocphala;B. americanus/Field / TP(G25)-Metamorphsand TP (G25)-J(4 or 6 mnths)T/99.2EtOH(NS) /(N/Y); NNS1 TP/0.8Lor20TP/20L;72 hrSexdev(L)B. amerR. sphenH. versi.Sexdev (J)B. amer.R. sphenH. versiSurv All7.55(M)0.8<0.92<329.524.6NS12430.923>29.5>24.6NSSex ratio for all endpointsMortality not specifiedResults on larvae appear to be same as reported inStorrs & Semlitsch 2008 / E103378; Results notdose responsive between treatments and time;Uncertainties about test design for juvenilesQualitative-Lower levelof confidence; seecomments for Storrs & Semlitsch 2008 / E103378Sullivan & Spence2003X.laevis /XenopusI(Adults) /TP(NF46-48)-MT/99ACTN(0.0032) /(Y/N); NSglass6 TP/0.5Lstartingvariedovertime;every3-4 daysMetamGroSurv40<2032032020>320↑Time to metamorphosis w/nitrate↓Mass at metamorphosis; no effecton metamorphosis for this studyReported mean mortality for entireatrazine only study (p value notreported); when atrazine w/nitrite,mortality sign. greater for nitrateChanging exposure conditions and alterations inthe number of replicates;Qualitative-Lower level of confidence based onchanging exposure conditions and alterations innumber of replicates18Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effectwith 320 μg/L atrazine than 0 μg/LnitrateTavera-Mendozaetal2002 / E60639X.laevis/Xenopus I/ TP(NG56)for 48 hT/99NA /(Y/NA);Nglass16 TP/15L; static(48hrexposure)SexdevSurv<18--18--↓testes size, spermatogonial nestsand nurse cellsReported no mortalities in control ortreatment; mortality not reported interms of NOAEC/LOAECNeed documentation of the measurementendpoints, elaboration on the ecological relevancyof the measurement endpoints, and clarification ofthe number of exposed animals; used individualanimals not replicates as experimental unitQualitative-Lower level of confidence based onneed for analysis and endpoint elaborationTavera-Mendozaetal2002 / E77830X. laevis/Xenopus I /TP(NF56) for48 hT /99NA /(Y/NA);Nglass16TP/15L;static (48hrexposure)SexdevSurv<18NS18NS↓primary oogonia↑secondaryoogonia↑atresiaSurvival not specified but based onsample size it appears to be 100%Only one dose; uncertaintyof the nature of themeasurement endpoints, and the relevancy of theendpoint to higher order measures of organismalfitness; used individual animals not replicates asexperimental unitQualitative-Lower level of confidence based onneed for analysis and endpoint elaborationVilleneuve et al.2003 / 45867708X. laevis/ / Afor 26, 43 or 47dT /NSNA /(Y/NA);NSfiberglass15 A/120L;50% every3 daysBio/MolSurv25 (N)-->25--Gonad and brain aromataseMortality ranged from 3-19% andwas not presented in terms ofNOAEC/LOAECLack of replication in some studies; observedvariability in endpoints; potential water qualityissuesInvalid for studies (male) due to no replication;Qualitative-Higher level of confidence inremaining study (female) due to higher variabilityin endpoint in controlWanet al. 2006 /E89626R. catesbeiana/Ward’s NaturalScience/ TP(10cm) for 96hrT /98 &F /48.5ACNT(NS);(Y/Y); NS(dilutionwateranalysed)glass0.8 g/L;staticSurv -- -- Technical96 hr LC50 > 16000Formulation96 hr LC50 > 480000 (232800 ai)Solvent concentration not reported; raw data notprovided, control mortality not reportedQualitative – Medium level of confidence basedon lack of summary data and no reported solventconcentrationWilliams &Semlitsch 2010 /E153825B.americanus&P. triseriata&H. versicolor/ Field / TP(G25)-MF /48.8NA /(Y/N); NGlass&plasticNS (1TP/1Lcontainer);72 hrMetamGro(allspecies)Surv1.25(M)1.251.25>1.25>1.251.25Time to metamorphosisMass% SurvivingLimited water quality reported; fill volume of testvessel not reported (1 TP /1L container)Qualitative- Lower level of confidence based onlimited water quality reported with a renewalperiod of 72 hrs and probably slightly higher thanrecommended loadingZayaet al. 2011a /E153847X.laevis/ NS(Nasco?) / TP(NF47)-NFT/NSNA /(Y/NA);NSglass4 TP/1L;3X/wkMetamGroBehav200 (N)25400400200>400Time to metamorphosisMass; fat bodiesFeedingPotential effects of stress during weighing andstaging during study; uncertainty about protocolfor feeding endpoint; variability in fat body and19Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect58or 62SexdevImm/infSurv400200400>400400>400Sex ratio (gross morphology)↑Activated caspase 3 immuno(+)cellsSurvivalliver size endpoints; limited water quality reportedQualitative-Lower level of confidence based onlimited water quality reported with a higher thanrecommended loading and a renewal period of 72hoursZayaet al2011b /E153848X.laevis/ NS(Nasco?) / TP(NF47)-NF58or 62T/NSNA /(Y/NA);NSglass4 TP/1L;3X/wkBio/MolSurv<400 (N)400400>400Changes in gene expression (actylCoA ehydrogenase andglucocorticoid receptor)Used organisms from Zayaet al2011a (E153847)etiology of the effectson gene expression reportedin this article are uncertain; potential bias insampling; limited water quality reportedQualitative-Lower level of confidence based onpotential biasing and limited water qualityreported with a higher than recommended loadingand a renewal period of 72 hoursCosmsBoone & BridgesBritton 2006 /E88290Hyla. versicolor/ Field / T(G25)- M (35d)F /40.8NA /(Y/NA);NSPE30 TP/1000LMetamGroSurv20 (N)2020>20>20>20Days to metamorphosisMassSurvivalLow power to detect differences;QualitativeBoone & James2003 / E81455R.SphenocephalaB. americanusA. maculatum& A. texanum /Field / LV for39-41 or 80 dF/40.8NA /(Y/NA);NSPE20 or 60TP/1000LMetamGroSurv197 &<197<197197>197 &197197>197↑time to metamorphosis for A.texanum; no effect other species↓Mass (all species)SurvivalLimited data to interpret atrazine only effects forsome endpoints/scenarios; lack of analyticalmeasurements in dilution waterQualitativeDeNoyelles 1989 /47543608R. catesbiana/NS/ Stage NSfor 805 dF /41 &T/97NA /(Y/NA);NSNS NSGroSurv<20 (N)20↓overall biomassBullfrogs immigrated to ponds-uneven distributionlikely; potential chemicals in ponds prior to testingunknownQualitativeDetenbecket al.1996 / E16839R. pipiens/Field / TP (NS)for 41 dF/ 85NA /(Y/NA);YWoodencagew/plastic20 TP/cage/floweringstreamMetamSurv25 (N)NS>25NS% adult appearance; hind limb lengthNot specifiedControl contamination; short acclimation periods;higher variability (lower power)Qualitative based on review for plantsDianaet al2000 /E59818H.versicolor/Field / TP tometamorphsT/NSACTN(0.04% )/(N/Y); Yplastic40 LV/90LMetamGroSurv2000202000>2000200>2000Time to metamorphosis↓Mass; SVL% SurvivalControl contamination; pre-metamorphosis weightand length were not determinedQualitative based on review for plantsDu Preezet al2008(lab and cosm) /E103271X laevis /Field/ TPto A (2 yrspost metam)T /NSMeOH(0.025) /(N/Y); NNS75frogs/2,500LMetamSexdevSurv24.824.824.8>24.8>24.8>24.8Time to metamorphosisGonadal development; sex ratio% SurvivalContinuation of Jooste 2005 (46965603); Tempconcerns; uncertainty about adding tadpoles atdifferent times effect on metamorphosis endpoint;20Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effecthigher occurance in testicular oocytes in controlsInvalid based on control oocytesJoosteet al2005 /MRID 46965603X.laevis / Field(Adults) / TP -MT/MeOH(0.025%) /(N/Y);NS24PE800 TP /1100LMetamSexdevSurv30.8 (M)30.8NS>30.8>30.8NSTime to metamorphosisTesticular oocytesPreliminary to DuPreez 2008 (E103271);Unusually long development times; feeding andtemperatures may have limited development.Higher incidence of testicular oocytes in controlsInvalid based on control oocytesLanglois 2010 /E120046R.pipiens /Field /TP(G27)-M(G42)F /(NS)NA /(Y/NA);YHDPE 0.1 g/LMetamSexdevBio/MolSurv<0.10.10.10.10.11.81.81.8↓Metamorphic success↑Sex ratio (female)↑and↓Brain eralph and tail doi3Total survivalControl contamination and atrazine detected inarea where organisms collected; uncertainty inbiochemical responses on gross endpoints;metamorphic success and sex ratio based on totalexposed, no replicate variability reportedQualitativeRelyea 2009 /E114296H.versicolor &R. pipiens / NS(collected aseggs) / TP-MT/99EtOH(0.003) /(Y/Y);NS25cattlewateringtanks9 TP/m2H. versMetamGroSurvR. pipiensMetamGroSurv6.4<6.46.46.46.46.4>6.46.4>6.4>6.4>6.4>6.4Time to metamorphosis↑MassSurvivalTime to metamorphosisMassSurvivalAtrazine concentrations were not monitoredthroughout the experiment, although an initialmeasured value was reported. Control tanks werenot analyzed for atrazine and the other ninepesticides tested. Additionally, it is unclear howmuch ethanol was added to atrazine-only treatmenttanks.QualitativeRohr and Crumine2005 / E117319R.sylvatica/Field / TP-MF /ACTN(0.00246)/ (Y/Y);NS26PE20 TP/10LMetamGroBehavSurv5050<50-->50>5050--Stage at test terminationMass↓% of hidingReported no dead tadpoles foundduring study; mortality not presentedin terms of NOAEC/LOAECAtrazine concentrations not monitored; unclearacclimation periods; prior exposure for tadpolesQualitativeRohret al. 2008a /E112910R.pipiens & R.clamitans /Field/ TP for 4wksT/98NS (Y/Y);NS27plastic65amphibs/800LImm/InfSurv<117117117117↓eosinophils↑trematode cycts (R.clam);↓Melanomacrophage↓% survival forR. pipiensand noUnknown solvent and concentration. Atrazineconcentrations were not monitored during test.Unknown if in algal communities similar at testinitiation .24Atrazine measured in treatment tanks; unclear if analyzed in control25Controls not analyzed; however, dilution water prior to addition to tanks was analyzed and found to be free of atrazine26Atrazine measured in treatment tanks; unclear if analyzed in control27Atrazine measured in treatment tanks; unclear if analyzed in con27trol21Citation /ECOTOX(E)/MRIDSpecies /Source /ExposurePeriod (stages& testduration)1TS2/ %aiSolvent(%)3/NC/SC(Y/N);MA inCNT(Y/N/NS)4Testvesselmaterial5Loading;RenewalPeriod6Reported Results7Indentified Uncertainties / Concerns andClassificationEndpointNOEC(μg/L)LOEC(μg/L)Effect&<117 &>117 effect forR. clamitansQualitative22 2. Summary of Amphibian Open Literature as of the 2007 SAP on Amphibians

Several of the open literature studies ( e.g ., Gibble et al . 2011 28 ; Koprivnikar 2010 29 ; Houck and Sessions 2006 30 ) examined potential effects of atrazine on varying parameters associated with immune response for aquati c-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 atra zine concentrations of 3 μg a.i./L. Additionally, leopard frog larvae exposed to 300 μg a.i./L exhi bited 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 a nd lymphocyte counts and reported

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

include information on the source and numb er of test animals; husbandry conditions were a major source of uncertainty as anim als did not appear to feed during the study period. In Haridon et al. 1993 31 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 stat istical analysis and used dimethly sulfoxide as a solvent and it wa s not clear if the study included a solvent

control.

In another study by Koprivnikar et al. 2007 32

, 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 in fection between atrazi ne groups and control cercariae. There was a significant differen ce in cyst abundance and intensity between control and 30 μg/L group exposed to control cercariae. The author s indicate that the presence of control cercariae affected tadpol e activity whereas atrazi ne concentration did not affect tadpole activity as we ll as no interaction was found between effects of cercariae and atrazine. However, there was only an eff ect when the tadpoles but not the cercariae were exposed to atrazine, therefore, there is uncertainty in the environmenta l relevance of the study. In addition, based on the study design, a control gr oup that was not exposed to

28 Gibble, R.E. and K.N. Baer. 2011. Effects of atrazine, ag ricultural 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 at razine 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

‐ 217023 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 2009 33 , researchers examined the effects of atrazine (10, 20 or 200 μg a.i./L) on the susceptibili ty 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 auth ors, significant inte ractive 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 inf ection rate nor did it significantly affect viral load. The study did not employ a solvent control a lthough methanol was used as a co-solvent; additionally, the study was conducted in pl astic containers.

Rohr et al . 2008 34 examined the effects of static 4-wk exposure to atrazi ne 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 Plagiorchi dae; snails were rotated among tanks every other day for "homogenization" purposes althoug h the snails were reported as not having shedded cercariae uniformly/consistently th roughout the study period. The study also reports on a survey of eighteen wetlands in the Broadleaf Forest Ecoregion of Minnesota to examine 240 "plausible pred ictors" of the abu ndance of larval tr ematodes in northern leopard frogs (R. pipiens). In 1999, the author s characterized the bi ological diversity of the study sites and obtained water, sedime nt and recently metamorphosed frog tissue samples for parasite quantific ation and pathology. Frogs (G osner Stage 46) were also examined for melanomacrophages and eosinophils . Results from the mesocosm studies indicated that atrazine significantly redu ced phytoplankton chlorophyll a concentrations while periphyton chlorophyll a concentrations were significantly increased. Snail populations in the atrazine tr eated tanks were also significantly higher (4x) than in controls. According the study authors, this reduction in phytopla nkton 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 atr azine-treated tanks (47.5% survival in the atrazine treatment group versus 70% survival in th e 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-treat ed mesocosms showed significant reductions in liver eosinophil

counts. R. clamitans in atrazine treated tanks ha d significantly more plagiorchid

33 Kerby, J.L and A. Storfer. 2009. Co mbined effects of atrazine and chlorp yrifos 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 su rvey, the authors state that atrazine combined with desethylatrazine (DEA) was th e 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 peri phyton) and by suppressing amphibian immune responses. Environmen tal conditions of the mesocosm studies

discussed in the article are not well describe d and whether the experimental units were roughly equivalent prio r to study initiation.

A study tested the interaction of emerging infe ctious diseases and atrazine in long-toed salamanders ( Ambystoma macrodactylum ) (Forson and Storfer 2006 35 ). The effects of

atrazine and virus was tested on mass and s nout-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 mort ality 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 2006 36 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 si gnificant differences on viral copy between any atrazine treatment group. Fo r atrazine only exposures, there were no effects on mass, SVL or larval period. Li mited water quality was reported and based on information provided, loading was assume d 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. Storfe r. 2006. Atrazine increases ranavirus susceptibility in the tiger salamander, Ambystoma tigrinum

. Ecol. App. 16(6): 2325-2332.25 In Kiesecker 2002 37 , wood frogs (Rana sylvatica) were exposed to atrazi ne and Ribeiroia sp or Telorchis sp cercariae a nd a study authors indi cate atrazine resulted in a decrease in eosinophils and an increase in the proportion of cercariae that encyst ed was observed at 3 and 30 μg/L. This controls in this study al so contained the cercariae and an additional

control that did not contain atrazine or cercariae to obser ved 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 trea ted 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 treatm ent groups, 0.01-10 μg/L, compared to the thioglycollate stimulated control. In add ition, at 21 μg/L, only treatment group, atrazine significantly diminished innate immune respons e when compared to frogs not exposed to atrazine but injected with th ioglycollate (an inflammatory mediator) as the number of peritoneal exudates cells was significantly decreased. A treatment group that did not

contain atrazine or was not tr eated further was not used in this study. While the study reported a change in white blood cells, these en dpoints were not clea rly and quantiatively linked to effects on survival, grow th or reproduction in this study.

Amphibian development was examined in several of the studies. In a study by Lenkowski and McLaughlin 2010 39 , 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 developm ent, the study authors assert that atrazine exposure re sults in an alteration of diffe rentiated tissues (cranial and muscle) and they hypothesize that this disr uption may be through m odulation of matrix metalloproteinases. The atrazine concentra tions tested in this study are high and the study relied on dimethylsulfoxide (DMS O). In another study by Lenkowski et al . 2010 40

, 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 significan tly 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 sup port this assertion. Similar to the previous study by Lenkowski, the vari ous treatment solutions contained DMSO

and treatment concentrations at which effect s are noted are relative ly high; additionally,

environmental conditions used in th e 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 at razine exposure disrupts matr ix 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 triadime fon 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 aromatas e gene expression in the brain or kidneygonadal complex of undifferentiated (NF Stag e 48) and differentiated (NF Stage 58)

larvae.

Williams and Semlitsch 2010 41 examined the effects of at razine exposure throughout the larval period to survival di fferences 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 form ulated 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 metamo rphosis or larval pe riod length at the

concentrations tested.

Storrs-Mendez and Semlitsch 2009 42 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 ra te of sexual differentiation were more susceptible to atrazine as larvae and metamor phs 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 comp letion of metamorphosis. For R. sphenocephala (long larval period with accelerated rate of gonadal devel opment, 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 diffe rences in sex ratio at 6 wk s 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 atrazi ne-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-treat ed and control Rana; however, ovotestes were observed in both contro ls 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 conclude d that a phase of intersex gonads is normal during leopard frog (rapid differentiation) sexual developmen t and that intersex gonads were found in juvenile stages of toads and treefrogs representing animals with slow and basic rates of gonadal differentiation. Treefr ogs exhibited underdeveloped testes in a limited number

41 Williams, B. K. and R. D. Semlitsch. 2010. Larval Responses of Three Midw estern Anurans to Chronic, Low-Dose Exposures of Four Herbicides. Arch. Environ. Cont am. Toxicol. 58: 819 - 827 (ECOTOX Ref. No. 153825)

42 Storrs-Méndez, S. I. and R. D. Semlitsch. 2009. Inte rsex Gonads in Frogs: Understanding the Time Course of Natural Development and Role of Endocri ne Disruptors. Journal of Experiment al 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 negativ e 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 concentrati ons ranges from 1.5 - 4 times higher than nominal. Depending on the species and sa mpling period, control animals appeared to reflect relatively skewed sex ra tios. The fluctuating sex ratio s in ethanol controls suggest that gonadal development was inconclusive pr ior 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 . 2011 a 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 atrazi ne 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 stai ning 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 hr s 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 animal s exposed to 400 μg a.i./L only one of the five studies exhibited signifi cantly reduced SVL relative to controls. There were no differences in metamorphic stages between c ontrols and animals tr eated 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 la rvae 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 fa t body and liver histology of atrazine-treated larvae and there were no differences in glyc ogen or lipid content of the livers; however, the authors noted that in one study, larvae expo sed 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. Whita ker, S. Kohler and C. F. Ide. 2011 a . 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 . 2009 44 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 determin e whether the toads pr eferred treated or untreated soil. A subgroup of the toads (f asted 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 an imals was recorded and toads were taken for analysis at 2, 4, 8, 12 and 24 hrs after exposur e. 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 la beled atrazine. In the soil preference study

measured atrazine levels in the treated so il 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 statisti cally significant preference for treated versus untreated soil. In the uptake and elim ination study, the high est percentage of radioactivity was in the in testine. According to th e study authors, the highest concentration was in the gall bladder and intestine at 8 hrs a lthough figures in the publication suggest that peak levels o ccurred at 12 hours in both tissues; peak concentrations in other tissues (i.e., liver, st omach and kidney) occurred at 2 hours. This study is seriously confounded by the absence of a solvent (ethanol) c ontrol, the reuse of previously tested animals (albeit 4 months pr evious) and uncertainty regarding the nature of radio-labeled residues ( i.e ., parent atrazine or a partic ular transformation product).

In Rohr et al . 2009 45 , tadpoles were evaluated to determ ine if exposed to atrazine for 4 days altered their perception (re sponses to) of risk of preda tion and parasitism. Previous exposure to atrazine did not si gnificantly affect responses fo r any of the cues. Cercariae alone and infected snail treatments significan tly increased tadpole activity (by number of segments crossed); but other treatments were not significantly different including previous exposure to atrazine. The st udy provides information on the survival of tadpoles to atrazine for 4 days, however, the results of the behavior al studies cannot be used for the risk assessment endpoints of su rvival, growth or reproduction as direct measures of these endpoints were not conduc ted (exposures to queues were for one hour only).

In mesocosm studies (Langlois et al . 2010 46 ) with wild-caught R. pipiens , larvae (Gosner Stage [GS] 25) were exposed through metamorphosis to nomi nal 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 wa s significantly lower for larv ae exposed to 1.8 μg a.i./L (66%) and EE2 (65%) compared to controls (76 – 79%) with signi ficantly 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. Pau li, 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:fema le; 1:1.14) was significan tly different (higher percentage of females) compared to controls (1:0.6). The se x 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 repor t 22% of the males in the EE2 group had testicular oocytes, no testic ular 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 treat ment. Atrazine did not affect aromatase (cyp19mRNA) expression in pre-metamorphi c larval (G34 and G45) brain tissue; expression of estrogen receptor al pha (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 atr azine 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 app ear to exhibit signi ficantly 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 alte red sex ratios/feminization. Also, although the authors assert that they pr esent 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 obs erved 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 (4 o C) water temperatures betw een the control and 1.8 μg a.i./L atrazine treatment. Also, there appear ed 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 2005 47
to determine whether a 4-wk static exposur e 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. A lthough 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. Atrazi ne concentrations were not monitored during the study and the study authors report that nom inal concentration we re likely higher due to heavy rainfall that occurred during th e study. Periphytic alg ae were significantly reduced (roughly 15% decrease in chlorophyll a) and water clarity was greater in

atrazine-treated mesocosms. Lower densit ities (roughly a 24% decr ease) of dipteran larvae ( Polypedilum ) were observed in atrazine-treat ed 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-t reated 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. Additi onally, atrazine treatment did not have a significant effect on final tadpole mass. According to the study authors, the direct effects of atrazine in reducing periphy ton (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 w ith the absence of a negative control are potential limitations in the study.

For Boone and James 2003 48 Atrazine exposur e at 200 μg/L, the only concentration tested, did not influence the mass, SVL, deve lopmental stage, or survival of spotted salamanders( Ambystoma maculatum) in outdoor a mesocosm study. For leopard frogs Rana sphenocephala , atrazine exposure did not signi ficantly influence survival. However, survival was significantly influe nced by an atrazine by population density

interaction where atrazine e xposure decreased survival in the high-density ponds but not the low-density ponds relative to the contro l ponds (treatments wit hout atrazine) at 200 μg/L. Mass significantly decrea sed with atrazine exposure co mpared 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 2009 49 mesocosm study, for leopard frog tadpoles ( R. pipiens ), there was not a significant difference between the atrazi ne treatment and the no-pesticide treatments (i.e. negative and ethanol control tanks) rega rding survival to metamorphosis, mass at metamorphosis, and time to metamor phosis. 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 metamorphos is 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 . 2009 50 , 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 metamorphos is. 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 mi xtures of pesticides at low concentrations affect aquatic communities. Oecologia 159(2): 363-376.

50 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: Non-monotonous acceleration of

the time to climax and de layed tail resorption. Aquatic Toxico logy. 91 (161-170). (ECOTOX Ref. No. 112588)31 10-, 14- and 21-day LC 50 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 LC 50 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 LC 50 values of 18270, 14430 and 12630 μg a.i./L, respectively. According to th e study authors, Stage 4 embryo s, 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 sens itive. None of the larvae in the 15,000 and 20000 μg a.i./L reached metamorphosis and onl y 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 curv e with 100 and 1000 μg a.i./L having faster times and the 5000 μg a.i./L having no differe nce 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 em bryos required de-jellying of the eggs and it is uncertain how this pr ocess affects exposure to the te st substance. Also, although the time required for 50% of the larvae to r each Stage 42 and/or complete metamorphosis was significantly different from controls for one or more of the atr azine treatment groups not affected by survival, there was no signi ficant difference between these groups for the

duration between when 50% of the test anim als reached Stage 42 and the completion of metamorphosis. Hyla versicolor tadpoles (Gosne r 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 tadpol es that initiated metamorphosis at 200 μg/L, but not 20 μg/L. However, while loading was within recommended limits, limited water quality wa s reported with a relatively long renewal period (50% renewal of solution weekly).

Xenopus laevis tadpoles were exposed to atr azine and tadpoles were staged weekly and upon complete metamorphosis, total length a nd 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 we re removed for histological analysis or biochemical analysis. . After metamorphosis, the sex ratio wa s 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. Acco rding to the study authors, evaluation of sex by gross morphology was corroborated by histological analysis. Time to metamorphosis was similar among treatment gr oups and there did not appear to be an

acceleration or inhibiti on of development.

51 LaFiandra, E.M, K.J. Babbitt, and S.A. Sower. 20 08. 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: 215-22632 In Allran and Karasov 2000 53 Rana pipiens larvae were exposed to atrazine and nitrate from Gosner stage 25 till metamorphosis. A ccording to the authors, atrazine did not affect percent metamorphosis, time to metamorphosis, percent survival, mass at metamorphosis or hematocrit at concentr ations up to 200 μg/L; while there was no consistent effect of atrazine on growth, th ere was significant atrazi ne 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 ma ss endpoints, statistical analyses were conducted on 50% of the organisms at test term ination and the reason fo r this is not clear.

In another study by Allran and Karasov 2001 54 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 da ta 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. 1998 55 , tiger salamanders larvae (Ambys toma tigrunum) exposed to 81.8 μg/L atrazine reached stage 4 during devel opment 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 compar ed 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 . 2010 56 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 Gosn er 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, se x ratio or growth (tot al body length) of the spotted marsh frog; however, testicular oocyt es 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, larv ae, 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). Ef fects 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 herb icide mixture. Chemosphere 78: 807 - 813. (ECOTOX Ref. No. 153638)33 A second paper by Zaya et al . 2011 b 57 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 . 2009 58 and Zaya et al. 2011 a ) 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 expos ure 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, gl utathione 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 ba sed on their previo us publication (Zaya et al

. 2011 a ), larvae were significantl y smaller within 72 hrs of exposure and at NF Stage 62 had depleted fat stores at th e 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 st udies examining ATP levels, there were no significant differe nces noted between control and atrazine-treated animals.

The authors conclude that base d on changes in gene expression, larvae exposed to 400 μg a.i./L experience an energy imbalance star ting early in exposure; however, they are uncertain regarding the etiol ogy of this energy imbalance. The article refers to Langerveld et al . 2009 to support claims that atrazine exposure at 400 μ g/L resulted in significant reductions in survival and gr owth (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 mo rtality of test animals in control and atrazine-treated groups was 63% and 57%, respectively. Th is further suggests that environmental conditions in the study were not sufficient to support animal survival. In studies where environmental conditions are poor and test an imals are struggling to survive, it is uncertain whether any meaningful relationshi ps 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 a nd excretion of atrazi ne and/or endocrine disruption is broad, and with resp ect to endocrine disruption, it is speculative. Given that the authors acknowledge that the glutathion [det oxification] 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 st udy results support the st atement that "clearly, acutely and chronically exposed tadpoles ar e compromised, decreasing their ability to survive the stresses of metamorphosis a nd/or to adequately support reproduction. Although the authors state that atrazine-exposur e 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 [s maller]; howeve r, it is unclear what that reduction in percent of body

57 Zaya, R. M., Z. Amini, A. S. Whitaker, and C. F. Ide. 2011 b 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 regul ate immune an growth-related functions in developing Xenopus laevis tadpoles. Environ. Res.109, 379–389.34 weight was associated with. Additionally, the authors state th at the rate of apoptosis was increased in NF Stage 62 larvae; how ever, the companion article by Zaya et al . 2011 a 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. 2011 59 examined the potential interacti on of temperature and atrazine exposure on growth, survival, hatching a nd 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 19 o C for 49 days until all embryos hatched or were dead. In a separa te experiment, larvae were exposed to the

same concentrations of at razine at 16, 19, 22 or 25 o C. In the 25 o C 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 a ffect larval survival; althoug h the authors indicated that no significant interaction was detected between temperature and timing of hatch, embryonic survival or larval survival, la rval mass was significantly higher at 19 o C compared to 13oc and embryos hatched sooner at 19 o C. Based on the larval studies, the study authors conclude that at razine exposure significantl y delayed metamorphosis and reduced mass at metamorphosis and that wh ile temperature was positively associated with developmental rate it did not significantl y affect size at metamo rphosis. Although

the author assert that there wa s a trend for atrazine to be a significant negative predictor of larval survival, this study did not detect any statistica lly 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 concentr ation delayed hatching and metamorphosis, reduced growth and decrease d 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 cont rol survival represent majo r limitations in this study.

Storrs and Kiesecker 2004 60 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 (sta ges 29-36) developmental stages. The results of the study showed counterintu itive 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 differe nces 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 signi ficant 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 effect s 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. 1054-105735 survivorship, relative to the control, were observed for late spring peepers, early American toads, and late wood frogs.

Rohr et al . 2003 61 examined 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 acco rding to the study author. Hunger stimulated a decrease in ref uge use and an increase in activity (based on regression; no significa nt difference detected at any le vel); however this response was least pronounced in the larvae exposed to at razine at 400 μg/L, possibly because these larvae were the most lethargic. The study au thors speculated that th e frog collection site may have been influenced by agricultural ac tivity including atrazine . In addition, DMSO was used as a solvent at a concentration gr eater than recommended for chronic testing.

In Rohr et al. 2004 62 , the combined effects of at razine, food limitation, and drying conditions on the survival, life history, a nd behavior of the streamside salamander ( Ambystoma barbouri ) from the embryo stage through metamorphosis were examined. Exposure to 400 μg/L atrazine decreased em bryo survival to Day 16 and increased time to hatching. However, most embryo mortalit y was associated with a white film covering the embryo, suggesting the presence of a f ungal pathogen. It is unknown whether the fungi caused or simply followed mortalit y. 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 accele rated 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 teste d. Frog collection site ma y 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 2005 63 . This study was a continuation of Rohr et al. 2004, and metamorphs used in this study which were obt ained from that study and were not further exposed to atrazine in this study. For ever y hour for 5 hours the mass of the metamorphs was recorded and they were observed fo r water-conserving be haviors (moving and pressed against dish and number of or ganisms huddled (when dished contained 3 metamorphs)). The same method description ab ove was used to retest the same animals a mean of 237 and 239 days after atrazine e xposure. According to the authors, the salamanders exposed to 40 and 400 μg/L e xhibited 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 Sublet hal 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 herb icide exposure increases salamander desiccation risk eight months later in a terrestrial environment. Environ. Toxicol. Chem . 24(5): 1253-125836 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 continua tion of Rohr et al. 2004, the same uncertainties as id entified in that st udy are expressed here . In addition, for this trial, the authors state that percen t 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 mort ality during exposure was ameliorated by density-depende nt survival after exposur e, 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 or ganism for this study is from Rohr et al. 2004, the same concerns apply to this study.

In a study by Olivier and Moon 2010 65 , 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 s oon 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 detect ed in the control samples at about 8% of the lowest test

concentration.

In another mesocosm study by Boone and Bridges-Britton 2006 67 , the single and

interactive effects of atra zine, carbaryl, and ammonium nitrate fertilizer on metamorphosis of tadpoles of the gray treefrog ( Hyla versicolor ) was examined. The

interaction of the atrazine and ammonium nitrat e 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: Break ing 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 spotte d 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): 3261-326537 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, th e 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 th at there was no effect on cl utch 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 uncertainti es identified in this study (from the 2007 SAP on amphibians) are of concern in this study. In that study

uncertainties identified included: unusu ally 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 a l. 1998 68 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), th erefore, 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 . 1980 69 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-recomm ended solvent (DMSO) and at higher than recommended concentration (1% in this a ssay as oppose to maximum rate of 0.05%) and greater loading than reco mmended. In Wan et al. 2006 70 , acute toxicity was examined using technical atrazine and a 48.5% formulation for the tadpole Rana catesbeiana (10 cm in length)

. The 96-hr LC 50 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: 52-5838 The genotoxic potential for Rana catesbeiana species was examined in Clements et al.

1997 71 and the study reported that all atrazine co ncentrations ≥ 4810 μg/L had significant

increases in DNA ratios relative to contro l and the linear corr elation between DNA damage and atrazine concentra tions 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 st udy was lacking in details on st udy design such as number of replicates and number of organism s exposed. In Freeman and Rayburn 2004 72 American toads ( Bufo americanus ) and Xenopus laevis tadpoles were exposed to atrazine and DNA from tadpoles was examined. According to th e 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. 2005 73 , American toads ( Bufo americanus ) were also exposed to atrazine and DNA from tadpoles was examine d. The total number of nuclei were not significantly different; however, control ta dpoles at significantly earlier stage and weighted less than tadpoles at 800 μg/L atrazi ne; 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 cont rols. In this study, water quality parameters were not reported and as stage and growth ar e 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 effect s on organogenesis. These studies reported effects at relatively hi gh 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 aut hors 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 a ll treatment groups. Axis malformati ons 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. Geno toxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell ge l 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, i ntestinal malformations and edemas were significantly increased in the 3.5, 10 and 35 mg/L atrazi ne concentrations. Also, according to the study authors, the cranial musc les 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 br ain 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 (correlati on coefficient 0.526, p=0.017) but was not observed for DEA. However, no negative control was included a nd 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 th e 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: 31-3840 3. INDIVIDUAL OPEN LITERATURE SUMM ARIES (in alphabetic order)

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

Chemical N ame: 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 wh ether atrazine and ni trate (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 concentratio ns (0, 5, 30 NO3-N/L) for a total of nine treatments each with 4 replicates with 20 la rvae per replicate (2.5 gal aquaria with 7-L solution). Dechlorinated water used as dilu tion 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 st age were measured. At metamorphosis (G42), frogs transferred to another aquaria wi th 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 di dn’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 ot her 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. Accord ing to the study authors, atrazine, nitrate and their intera ction had no significant effect on percent metamorphosis, time to metamorphosis, pe rcent 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 atrazi ne-by-nitrate-by time effect (p=0.0107) (Figure 1). There was also no consistent eff ect of atrazine or nitrate on development; the authors indicated a significant atrazine-by- nitrate-by-time effect was observed and clutches differed significan tly 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 nor thern leopard frog, however there are some uncertainties in the repor ting 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 da ta set had been included. Although the study author reported that there was no consistent effect on growth for atra zine (Table 1) they did indicate a significant atra zine by time effect; however th e authors did not state for which concentration at which time poin ts. 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 potenti al different stages affecting the growth

parameters.

Data on survival is not pres ented, therefore, control surv ival is not known. The authors

state that more than 90% of the animals re ached 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 clut ches and indiscriminately distributing larvae. It is not known why the clutches were kept se parate 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 wh ich 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 hi story is not reported. Ch lorine levels in the dechlorinated water are not repor ted as the analysis for potenti al 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 N ame: 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. To xicol 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 ve ntiliation 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 (D ay6) and post hatch effects including mortality, deformity, a nd 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 ventilat ion (breaths/minute) was significantly (p<0.05) at 4.3 mg/L a nd 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 a nd 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 insi ght 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 le vels 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, ERB345 3.3. Birge, W. J., J. A. Black and R. A. Kuehne. 1980

Chemical N ame: 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 Hatc hery, Frankfort Kentuc ky or obtained from Mogul-Ed, Oshkosh WI or Charles Sullivan, Nas hville TN. Atrazine was tested using a 80% wettable powder (formulation not stated) an d test solutions were confirmed daily. Tests were conducted under flow-through c onditions, 2.5 hr/turnover. Embryos were

tested at 30 minutes after fert ilization to 2-6 hours postspa wning. Sample sizes per test concentration ranged from 50-130 eggs and eggs were examined daily and dead embryos removed. Temperature, dissolved oxygen, wa ter hardness, pH, and specific conductivity

was monitored in the test water. Percent survival of normal orga nisms (free of gross teratic defects) was determined at hatch and 4 days post hatch.

LC50 results are presented in table below. Th ese values represent organisms that died as well as organisms with gr oss teratogenic effects.46 Description of Use in Document: Qualitative.

Rationale for Use: ,Provides information about the te ratogenic potential of atrazine on amphibian embryos and young larvae.

Limitations of the Study: Solvent (if used) was not iden tified. As formulation used, uncertainty in source of t oxicity (technical atrazine, inert ingredient or some combination). LC50s were not based on mort ality per se, but on abnormalities that would reportedly preclude survival under natural conditions.

Reviewer: Amy Blankinship, ERB347 3.4. Boone, M.D. and C.M. Bridges-Britton. 2006

Chemical N ame: Atrazine

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 88290

Boone, M.D. and C.M. Bridges-Britt on. 2006. Examining multiple sublethal contaminants on the gray treefrog (Hyla versicol or): effects of an insecticide, herbicide

and fertilizer. Environ. T oxicol. 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 interac tive effects of atra zine, carbaryl, and ammonium nitrate fertilizer on metamorphos is of tadpoles of the gray treefrog ( Hyla

versicolor ). Tadpoles were reared in mesoco sms from hatching through metamorphosis and were exposed to the presence or absence of the three contaminants. The results of the study indicated that the pr esence of multiple, subletha l chemical stressors with different modes of action may not be more detr imental 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 polyethyle ne ponds with a screen mesh lid. Three factors used in the study (carba ryl (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 period ically measured and metamorphs removed from ponds daily and held in laboratory until tail resorption at which time they were weighed. Study termin ated 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 nitr ate 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 effect s from atrazine and fertilizer

interaction but there are uncertainties in study.

Limitations of the Study: Low power to detect differences.

Reviewer: Amy Blankinship, ERB349 3.5. Boone, M. D. and S. M. James. 2003

Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Boone, M. D. and S. M. James. 2003. Interact ions of an insectic ide, 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 rega rding effects of atr azine 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 a nd carbaryl (study desi gn included individual and combined exposure treatments) on phytopl ankton and four amphibian species in outdoor mesocosms. Tadpole density a nd 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 al so 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 Foru m 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 he ld until all larvae were free-swimming. Clutches were mixed within each spec ies to homogenize genetic variation.50 The aquatic communities were prepared by f illing 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 natura l 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 na tural conditions. In order to protect the test systems, the cattle pond ta nks were located inside a fe nced 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), carba ryl 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 w ithout stirring. Uncontaminated pond water (5 L) was

added in the same manner to each control pond. The herbic ide/insecticide treatment was
added between 1000 and 1100 hours Central St andard 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 hour s post-treatment. Samples from each pond were combined, and an aliquot (20 mL) wa s refrigerated for analysis. Ponds with only atrazine were sampled (triplicate 2-L al iquots 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-liv es 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 leng th (SVL, salamander larvae only) and pond survival was determined. Data were analyzed for effects of carbaryl, atrazine, density (for tadpoles) and all inte ractions on mass, developmental stage and SVL using multivariate analysis of covariance (M ANCOVA) for ponds with both species. Univariate analysis of covariance (ANCOVA) was used for determining which responses were the strongest contributors to the multivaria te 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 propor tion data, log transformed mass and SVL, and used rank procedure (PROC RANK) on deve lopmental stage data before analyses. Conservative pair-wise compar isons for significant main e ffects were performed using Scheffé’s multiple comparison tests.

For Experiment II, the effects of hydrope riod, atrazine, and carbaryl on amphibians reared through metamorphosis were studied by a fully crossed design with four replicates (32 ponds) where hydroperiod was either consta nt 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 firs t experiment indicated that competition between anurans and salamande rs had no effect. Carbaryl (liquid Sevin, 22.5% carbaryl) and atrazine (liquid Aa trex, 40.8% atrazine) were added. The

herbicide/insecticide treatm ent 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 analys is. Ponds with only atrazine were sampled (triplicate 2-L aliquots from each of the thre e 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 multivariat e 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 M ANCOVA; survival was the covariant. ANCOVA was used for determining which respon ses 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 incuba ted with 15 mL of

neutralized 90% acetone in the dark at 5°C for 24 hours. Chlorophyll was measured using fluorometry. Water samples were take n 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 e ffects of time, carbaryl, and atrazine for both experiments in addition to density (Experi ment I) or hydroperiod (Experiment II) using

ANCOVA.

Results:

Experiment I: effects of competition, atra zine, and carbaryl on larval amphibians

Atrazine exposure at 200 ug/L did not influen ce the mass, SVL, developmental stage, or survival of spotted salamanders. There was a significant interact ion of atrazine and carbaryl on SVL that the authors attributed to atrazine acting as an ameliorating agent for the carbaryl treatments. Similar observa tions occurred on body mass and developmental stage, although the interacti on was not significant (P>0.05). For leopard frogs, atrazine exposure did not significantly influence survival. However, survival was significantly52 influenced by an atrazine by population de nsity interaction where atrazine exposure

decreased survival in the hi gh-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 e xposure; mass significantly decreased with atrazine exposure compared to the contro ls (P=0.0052). Results of the competitive

controls indicated that no difference occurr ed 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 indi cated that atrazine exposure and hydroperiod (individually and their interaction) si gnificantly affected mass and time to metamorphosis. The effect of atrazine e xposure was attributed to increasing time to metamorphosis. Time and mass to metamo rphosis were affected by the atrazinehydroperiod interaction, resulti ng in longer larval periods in constant hydroperiods and smaller mass at metamorphosis in drying hydrop eriods. 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 si gnificant effect on chlorophyll content. After 12 days of exposure, significantly lower ch lorophyll 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; F 1,24 = 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 c ontained carbaryl). The aut hors 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 wa s 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 signifi cant effects on chlorophyll at the end of the study (P = 0.62). However, chlorophyll content was only m easured after 42 days of exposure in experiment II. In experiment I, chlorophy ll content had already r ecovered after 25 days of exposure so it is reasonable to assume that any effect in experime nt II may have been missed due to insufficient sampling. No figures were provided which illustrated the chlorophyll content th rough 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 at razine-by-carbaryl int eraction over time from Experiment I. Numbers rep resent 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 200 1 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 wh ich chlorophyll content was measured in

order to examine effects of atrazine on amphibians via dimi nishing their food source, the aquatic plant life. The study au thors were looking for statisti cal 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 arti cle 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 concen tration of atrazine (200 ug atrazine/L). Atrazine was applied as part of a formulated product (Aatre x). It is unknown if there were any effects due to other i ngredients in th e formulation.

There was an acclimation period but its dur ation 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 meso cosms. It is unclear if the pond water used for adding the chemical tr eatment had phytoplankton in it and if so

how much. Based on the mean chlo rophyll concentrations presented in Figure 1

, chlorophyll concentrations appear similar prior to and a few da ys after application. It would be helpful to know more about the me thodology 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 obser vation of reduced chlorophyll relative to controls at Day 12; however, the reduction was somewhat s light and transient (effect observed at Day 12, but not at the next measurement on Day 25). In addition, there was

no indication of statistic al significance at Day 12 (i.e., if atrazine-o nly treatments were statistically analyzed separate ly) 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 th e 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 si ngle effect of atrazine. No statistics or information about variation among replicates was provided regard ing atrazine-onl y treatments. In addition, lake water was used, and there was no indica tion 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, EPA57 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 eval uate 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 (s tage 25) and prometamorphosis (stage 38-39).

Methods

Adult South American toads ( Rhinella arenarum ) were obtained from a local provider. Ovulation induced by injecting homologous hypo physis and oocytes fertilized in vitro using fresh sperm. Embryos maintained unt il 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. AMPHIT OX solution (AS) used as dilution water

solution.

Studies conducted using stage 4 embryos (jel ly coat removed prior to exposure) and Gosner stage 25 (premetamorphosis larvae) we re conducted by placing 10 eggs or larvae into each of three replicates glass Petri di shes (containing 40 mL of test solution) and monitoring them for survival and malformati ons 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 boile d swiss chard was added. The study using embryos was repeated twice with a distinct pair of parents for each study. The study58 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 st age 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 pa ir of parents.

Results from the control and solvent contro l 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 l ogistic 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. Fo r 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 <d etection 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 re ported that 10 days of expos ure was needed to observe a significant decrease in survival whereas fo r larvae exposed at stage 25, significant

decrease in survival occurre d 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 ind icates embryos presenting an early version of the given stage.

In the study using stage 25 larvae and low levels of atrazine, the aut hors 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 de veloped 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 observe d 2-3 days after exposure. For the larvae exposed at stage 38-39, no larvae survived at 15 and 20 mg/L with le ss 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 stag es tested, stage 25 wa s the most sensitive stage, however, once embryos exposed at stag e 4 reached stage 25, le thality was greater over next 10 days as stage 25 compared to le thality 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 accelerat ed 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; howev er, 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. Gi ven 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 examine d. They also state th at 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 di fference in low level toxicity in this study61 compared to others need to be examined to de termine 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 valu es), but invalid for use in comparisons for completion of metamorphosis.

Rationale for Use:

The study provides LC50 values for a toad spec ies (although not native to U.S.) that can provide insight into acute and chronic mort ality 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 to ads 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 repor t levels of potential contamin ants 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-j ellied prior to exposure. The reviewer

assumes this was conducted for ease of di stribution and potentially more uniform exposure. However, this process of re moving the jelly may not reflect exposure

conditions in the environment and make th e embryos more suscep tible 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 st udy makes the ability

to make a definitive conclusion about the e ffects 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 gr aphs indicate that the differenc e 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, base d 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 repe ated twice or four times, respectively. However, using organisms from a single parental source li mits 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 atr azine on larvae. However, the use of a single clutch may provide more consis tent 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 EC 50 values instead of the results at test

termination. The authors conduct the anal ysis of time to stag e 42 and complete62 metamorphosis for 50% of the organisms but th en discuss the environmental relevance in context of a 80 to 90% metamorphosis with out conducting statistical analysis. The reasoning for conducting the an alysis 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 atrazi ne significantly accelerated metamorphosis and had a nonmonotonic dose-response (U sh aped) curve; however, give n 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 statemen t. 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 treatm ent groups not affected by survival, there was no significant difference between the treatm ent groups for the duration between when 50% reached stage 42 and co mplete 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 applicable63 3.7. Brodkin, M.A., H. Madhoun, M. Rameswaran, and I. Vatnick. 2007. Open Liter ature 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). E nviron. Toxicol. Chem. 26 (1):80-84. ECOTOX 103351

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 Nort h 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 gr oup only exposed to TN, and a group that was only injected with RN with the last two not cont aining atrazine. In the second experiment, 36 frogs were allocate d into six treatments (n=6/treatment): atrazine concentrations of 0.01, 0.1, 1.0 and 10 μg/L which all receiv ed thioglycollate stimulation (TA), one control group that only received injection with Ringer solution (RN) and one group that only re ceived thioglycollate stimul ation (TN). The frogs were maintained individually in Tupperware contai ners with 500mL solution at 25°C. Frogs were injected on seventh day with Ringer so lution or thioglycollate and euthanized on day 8 and a peritoneal lavage was performed with Ringer so lution. 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 ei ther nonphagocytic or highly phagocytic.

Results

In experiment #1, the authors state that atra zine at 21 μg/L significantly diminished innate immune response when compared to fr ogs not exposed to atrazine but injected with thioglycollate (an inflammatory mediator ) as the number of peritoneal exudates cells64 was significantly decreased. In experiment #2, all atrazine treatme nts has significantly decreased number of peritoneal exudates cell s 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 solu tion 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 gr oups, 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 treatmen t even if further comparison to the treatment groups may not be wholly adequate. While the study re ported a change in white blood cells, these endpoints were not clearly and quantiatively linked to eff ects on survival, growth or reproduction in this study. Frogs were cult ured using Styrofoam and maintained in plastic containers during the te st and these materials are no t 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 applicable66 3.8. Choung, C.B, R.V. Hyne, R.M. Mann, M.M. Stevens, G.C. Hose. 2011.

Open Liter ature 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 focu s 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 be tween 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 defi nitive 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, ag ed Sydney Australia ta p water) control and a mixture of 25 and 10 μg/L atrazine and te rbufos sulfone, respec tively, for 10 weeks to encompass complete metamorphosis complete ly. 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:d ark period. Test solutions renewed twice weekly along with measured te st concentrations (duplicate samples) and environmental quality (dissolved oxygen, elec trical conductivity, pH, and temperature). Each week each tadpole was photographed to examine visual inspection of physical abnormalities and measure snout-vent length (S VL) and tadpole stage. When tadpoles reached stage 42, they were transferred to a nother test vessel containing small amount of test solution until tail resorpbtion was comp lete at which time SVL and weight were

measured.

Results The results presented will focus on the defini tive test. There we re three mortalities overall, one in terbufos and two in comb ined, with all other tadpoles completing67 metamorphosis. There was no significant difference in survival, SVL, mass, or metamorphosis between the control and atra zine-only group (Figure 3). There was a significant delay in development in the comb ined group as well as the terbufos only group. The study authors state that atrazine did not interact synergistic ally with terbufos sulfone or on growth and development; alt hough there was some indication of accelerated metamorphosis in the pilot study this should be interpreted with caution as the definitive

test no differences were obs erved [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 atrazi ne 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 the68 water-only control, atrazine, terbufos sulfone (T. sulfone) and the mixture of atrazine andterbufos sulfone (Mix) compared to the wateronly control. Treatments th at 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 statisti cal test is not know n. It is also not known what effect lowering the α level had on the results.

Given that there was only one atrazine concentr ation 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 applicable69 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 alkali ne 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 polypropyl ene containers containing dech lorinated 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 da rk to formulations (AAtrex Nine-O (atrazine); Dual-960E (m etalochlor), Roundup (glyphosate), Sencor500F (metribuzin), and Amsol (2,4-D amine )), methy methanesulfonate (MMS) or

dechlorinated water in for 24 hours in 1L beak ers containing 500 mL test solution. Test

concentrations for the herbic ides included the maximum surv ival 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 pr imary 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 sign ificant 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 calcu lated]. The linear correlation between DNA damage and AAtrex concentration was modera te 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 stat es procedure described in Ralph et al 1996 however, it appears to be for DNA assay not te st design [Ralph S., Petras M., Pandrangi, R., Vrzoc, M. 1996. Alkaline single cell ge l (comet) assay and genotoxicity monitoring using two species of tadpoles. Environ. Mol Mutagen . 28:112-120.]. Mortality not reported and with different number of animal s used in the assay for each concentration and no details on test design, the reviewer canno t discern mortality results. Water quality parameters not reported. Percent active ingr edient not reported, reviewer assumed based on available label (88.2% a.i.). As the expos ure was to a formulation, it is unknown if the observed toxicity was due to technical atrazi ne, an ingredient(s) in the formulation or

some combination. Based on the concentratio ns tested, the two highest concentrations tested appeared to be above the water sol ubility of atrazine (ca. 33 mg/L). While it provides some information on DNA damage, th e results are not directly correlated to survival, growth or repr oduction 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., Sc haeffer, D. J., Beckmen, K. B., and Beasley, V. R. (2000).

Chemical N ame: Atrazine (Amphibian Microcosm Study)

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 59818

Diana, S. G., Resetarits, W. J. Jr., Schaeffe r, D. J., Beckmen, K. B., and Beasley, V. R. (2000). Effects of Atrazine on Amphibia n 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 e ffects of atrazine on survival, mass, and length at metamorphosis, and days to meta morphosis of larval gray tree frogs ( Hyla

versicolor ) inhabiting artificial pond microcosms and to assess interrelationships among these parameters and DO concentrations, wa ter pH, and estimates of phytoplankton, periphyton, and macrophyte biomass.

Artifical pond microcosms (16 plastic wadi ng pools; 1.22-m diameter; arranged in 4x4 array on gravel pad) with 90 L pond wate r (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 concentrat ions declined sli ghtly during the 1 st 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 de nsities in the 200 and 2000 ppb groups increased significantly a bove control during rebound period. Over course of study (~40 days), ch lorophyll 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 retu rned to control values within the following 16 d. The DO72 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 signi ficant treatment-related differences were

detected for survival rate.

The decrease in amphibian length and we ight 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 f ood. However, atrazine-resistant species occurring in presence of c ontinued 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 e ffort at gill respiration, at the expense of feeding, when held in water with low DO.

Based on observed decreases in length and ma ss at metamorphosis, and decreases in pH, DO, and macrophyte biomass, it is suggested th at 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 vesse ls and therefore potential contaminants (leaching from pool) may have been present.73 Atrazine was measured immediately followi ng 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 concentr ation which is the level where significant

effects were observed.

Reviewer: Amy Blankinship, Chemist, ERB3, OPP74 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 Liter ature 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. Re production, larval growth, and reproduction development in African clawed frogs (X enopus laevis) exposed to atrazine. Chemosphere 71:546-552.

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 multigene ration with breeding evaluated from frogs exposed to atrazine with effects on number of eggs released, hatching success, and developmen t of F2 individuals monitored.

This study is a continuation of the co sm 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 pr ocessed and evaluated for gross pathology and histopathology.

According to the authors the size of the fema les influenced the clutch size with large differences in number of eggs. Also, the au thors 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):

INVALID75 Rationale for Use:

This study was a continuation of another st udy (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, ha tching 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 applicable76 3.12. Forson, D. and Storfer, A. (2006).

Chemical N ame: 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 c ontaminants, such as atrazine, may have suppressive effects on the amphibian immune sy stem, 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 10 3.5 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 iri dovirus 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 infectiv ity. ATV transmission was confirmed, although infection rates were lower than expected, c onsistent with the theory predicting lower pathogen transmission to nonnative hosts. La rvae exposed to both at razine 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 leve l (184 ppb) accelerated metamorphosis and reduced mass and SVL at metamorphosis rela tive 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 concentr ations initiate metamorphosis at a smaller size, with potential negative consequences to fitness. Larger size at metamorphosis is correlated with higher survival to matur ity and reduced time to maturity, thereby increasing fitness relative to smaller indivi duals [34-36 in paper]. Smaller size at metamorphosis may be a fitness cost resul ting from high-level atrazine exposure. Lighter, smaller animals have reduced terres trial locomotor performance and, therefore,77 reduced ability to avoid predators or capture prey [35,36]. Smaller, newly metamorphosed adults also tend to have w eakened 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 to tal of 160 individuals). Water was changed every 3 days.

Description of Use in Document: Qualitatively

Rationale for Use: Provided information on interac tion 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 a nd AZT). Water quality parameters, other than temperature, were not reported. Revi ewer 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, ERB378 3.13. Forson, D. and Storfer, A. (2006).

Chemical N ame: 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 c ontaminants, such as atrazine, may have suppressive effects on the amphibian immune sy stem, 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 10 3.5 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 iri dovirus 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 infectiv ity. ATV transmission was confirmed, although infection rates were lower than expected, c onsistent with the theory predicting lower pathogen transmission to nonnative hosts. La rvae exposed to both at razine 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 leve l (184 ppb) accelerated metamorphosis and reduced mass and SVL at metamorphosis rela tive 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 concentr ations initiate metamorphosis at a smaller size, with potential negative consequences to fitness. Larger size at metamorphosis is correlated with higher survival to matur ity and reduced time to maturity, thereby increasing fitness relative to smaller indivi duals [34-36 in paper]. Smaller size at metamorphosis may be a fitness cost resul ting from high-level atrazine exposure.79 Lighter, smaller animals have reduced terres trial locomotor performance and, therefore, reduced ability to avoid predators or capture prey [35,36]. Smaller, newly metamorphosed adults also tend to have w eakened 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 to tal of 160 individuals). Water was changed every 3 days.

Description of Use in Document: Qualitatively

Rationale for Use: Provided information on interac tion 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 a nd AZT). Water quality parameters, other than temperature, were not reported. Revi ewer assumed loading to be 1 larvae/500mL water which is lower than 1 tadpole or larv ae/1L recommended from 2007 SAP. Limited water quality data reported.

Reviewer: Amy Blankinship, ERB380 3.14. Fort, D. J., R. L. Rogers, J.H. Thomas, B.O. Buzzard, A. M. Noll and C.D. Spaulding. 2004

Chemical N ame: 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 espe cially in solvent choi ce and concentration.

Limitations of the Study: Water quality parameters not reported. DMSO was used at

1% and DMSO is not a recommended solv ent the recommended solvent concentration for acute assays is 0.05%.

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, Office of Pesticide Programs81 3.15. Freeman J.J. and A.L. Rayburn. 2004

Chemical N ame: Atrazine

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 78644 Freeman J.J. and A.L. Rayburn. 2004. In vivo ge notoxicity 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 overwint ered 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 atrazi ne concentrations of 0, 250, 500, 1000, 5000 and 10000 ppb for three weeks. Both tadpoles pr ocessed 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 tadpol es exposed to 0 and 800 ppb atrazine for 3 weeks after which they were processed for DN A analysis. In the second experiment, X. laevis tadpoles were processed at 3, 4, a nd 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 cont rol represented. Test vessel material and loading not reported. Survival not reported. The numbe r of replicates per treatment is not specified. Th e 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 Programs82 3.16. Freeman J.L. N. Beccue and A.L. Rayburn. 2005

Chemical N ame: 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 larv ae 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 adu lts 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 concen trations 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 af ter which they were staged, weighed and processed. This experiment was replicated three times. Ther e 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 obs erved and total number of nuclei were not significantly different; however, control ta dpoles 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 afte r 3 weeks, no differen ce in total number of nuclei was observed and weight was not signi ficantly 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 inform ation 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 fo r 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 cont rol represented. Test vessel material and loading not reported. Survival not reported, however based on sample sizes provided it appears it was 100%. As tadpol e 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, th e study authors accounted this in the study.

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, Office of Pesticide Programs84 3.17. Gibble, R.E. and K.N. Baer. 2011

Open Liter ature 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 investig ate the influence of common environmental pollutants on the production and in vitro bi oactivity of protectiv e peptides in an amphibian model ( X. laevis ).

Methods

The test materials used were technical gr ade and formulated at razine (ATR) with surfactant, agricultural runoff containing a mixt ure of pesticides, fiel ds treated with or seeds planted with a variety of chemi cals: paraquat, captan, diazinon, lindane, smetolachlor, and atrazine. Also tested were treated sewage treatment plant and municipal

landfill effluents. This review will focus on th e 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 con centrations of 0, 0 (DMSO, 0.0173% [reviewer calculated]), 0.1, 1, 10, 100 and 1000 μg/L unde r flow-through condi tions with two replicates for each treatment group. Concen trations were measured weekly from alternate reps. Tadpoles (NF 49/50) were al so exposed in a semi-static system (halfvolume renewals 3 times weekly) to the atr azine formulation AAtrex® 4L (40.8% ATR) mixed with Top Surf® surfact ant at atrazine concentra tions 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 fr om Bayou Desiard was used as dilution water; this water is a drinking water source and wate r quality parameters are monitored monthly. Larvae were exposed till completion of me tamorphosis (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 metamorphi c animals and analyzed for protein content and bioactivity. Antimicrobial secretions were induced by subcutaneous injection of85 norepinephrine bitartrate into the right dor sal lymph sac of post metamorphic animals, secretions pooled from 5 animals, a nd 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 tec hnical atrazine study were 79 to 105% of nominal, except for the 0.1 μg/L group wh ich 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. Accordin g to the study, in th e agricultural runoff samples, atrazine concentrations were initia lly 1.0 μg/L but increased to a maximum of 7.4 μg/L over ca. 8 weeks (1-2 weeks following second atrazine applic ation). There were no effects on survival, growth or development (data not presented). There were also no differences in total protein or slope betw een technical atrazine concentrations and controls as well as no signifi cant trend in total protein. For the atrazine formulation,

ANOVA detected difference in total protein, but Tukey’s pos t-hoc test did not; however regression analysis indicated si gnificant 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 e ndpoints 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, th e reviewer cannot eval uate the study authors results therefore large uncertainty for these e ndpoints. In addition, fo r the technical grade atrazine study DMSO was used as the solv ent 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 di d 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 numb er of organisms at each replicate for the AAtrex formulation study was not reported. Th e study did not report if the feed was free of potential contaminants. Atrazine was m easured in the solvent control (0.16 ppb) in which the author states may have been presen t for up to 48 hours. Also, the test vessels

were not described.

The results for the peptide assays and water quality characteristics on ly report one value for the 0 μg/L concentration. Therefore, it appears that the results were pooled for the negative and solvent controls fo r 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 an d the report does not explain why this group is so high.

For the peptide bioactivity, the study reports th at 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 replic ates for the technical atrazine study with no test design reported for the fo rmulation. Therefore, it a ppears that for this endpoint, groups of animals were treated as the experi mental unit and not the tanks. The peptide bioactivity assay was conducted by inducing antim icrobial secretions in the animals with a subcutaneous injection, theref ore there is uncertainty in th e 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 applicable87 3.18. Gunderson, M.P, N. Veldhoen, R.C. Skirrow, M.K. Macnab, W. Ding, G. van Aggelen, and C.C. Helbing. 2011

Open Liter ature 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 Ag gelen, and C.C. Helbing. 2011. Effect of low dose exposure to the herbicide atrazi ne 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 stea dy-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 (17 α -ethynylestradiol) and 1 and 100 μg/L DEA or atrazine for 48 hours; all treatments were prepared using isopropyl al cohol with a solvent concentration of 0.001%

and a solvent control was incl uded. Each treatment group had 5 tadpoles per replicate (20 gal glass aquariums with 50L well water; ave. loading 0.6 3 g/L). Dissolved oxygen, temperature and pH recorded at test initiation a nd termination. Tadpoles lost weight during the 48 hour exposure peri od however it as not significant between treatments. After 48 hours, tadpoles were euthanized a nd whole brains isolat ed and stored. Total

RNA was isolated from whole brain tissu e and cDNA synthesis and development of species-specific QPCR primers and assays conducted.

Results

The isolated partial bullfr og CYP19 cDNA sequence showed >90% putative amino acid sequence similarity across four anuran specie s; ca. 89% similarity with two salamander88 species and 81% similarity with two fish species. The study authors

indicate that there was a trend towards a decrease in CYP19 transcript levelsin 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 tadpol es 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 show n 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 reco mmended solvent (ASTM) . While the report indicates they were measured, results of the water quality pa rameters 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 applicable90 3.19. Hayes, T. B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. Adame, E. Chan, D. Buchol z, 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. Nazi r, 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 (phe notypic female) ZZ males plus ZW (genotypic/phenotypic) females and the resulting ZZ male larvae we re exposed to atrazi ne at 2.5 μg/L from hatching through metamorphosis and for a peri od of up to 3 years post-metamorphosis. According to the study, atrazine-exposed males were both demasculinized and completely feminized as adults; 10% of th e ZZ males (4 out of 40) developed into functional phenotypic female s/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 testoste rone, decreased breeding gland size, “demasculinized/feminized” laryngeal de velopment, suppressed mating behavior, reduced spermatogenesis, and decreased fertil ity relative to a solvent (ethanol) control

males.

Sex-reversed (ZZ) males (phenotypic fe males) were confirmed by absence of DNAbinding domain W (DM-W) mark er 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 ther e are 2 treatments (atrazine and ethanol control) with 9 tanks per treatment and 30 tadpoles pe r tank. Therefore, there are 270 animals per treatment (atrazine and ethanol control). An imals from three of the 9 tanks (representing 90 animals) from each treatment were sacrif iced at metamorphosis; however, according

to Figure S1 (reproduced below) of the Supporting In formation 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 per91 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) condi tions in fiberglass tanks at 22 o 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 obta ined 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 ta nks (46x25x20 cm; 23L) and raised singly (for fertilization experiments) or in groups of 4 (4 breedi ng 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 in volved in the study.

Atrazine levels in both treatment groups we re 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 extr acted from gonads of the two atrazinetreated females that were eu thanized, 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 fe males (hCG injected). The next day, eggs were collected, aerated, and allowed to devel op for 72 hrs. Fertilit y was determined by counting the number of devel oped embryos (NF stages 14 - 34). The study was repeated in December 2008 with 5 animals from each gr oup (atrazine and ethanol control). The animals from both these analyses were th e same ones used for histological and morphometric analyses. According to the st udy, atrazine-exposed males had significantly lower fertility rates (measured by the propor tion 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 were92 typically filled with mature spermatozoa bund les, whereas the majority of tubules in atrazine-exposed males lack ed mature sperm bundles and were nearly empty with secondary spermatocytes confined to the peri phery of the tubule. Additionally, atrazine treatment altered the physica l 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 follo wing day, and amplectant pairs were observed. Blood samples were then collected from pa irs 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 light ing 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 pres ence of females] when compared to atrazineexposed males. Control males (11/16) ou t-competed atrazine-exposed males for amplexus with females; however, two atrazi ne-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 st atistical analyses repo rted in the paper. For at least some analyses, judgment sampli ng 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 larv al exposure conditions that have been previously reviewed by EPA and FIFRA SAP and deemed capable of introducing serious confounding effects on the study. Additionall y, although the study “rearing program” is clearly depicted in the supplemental inform ation referred to in the main publication, reported sample sizes associated with various measurement parameters are not consistent with the rearing program depicting the study tr eatments and replicates within treatment. In general, and under the conditions tested in this study, atrazine e xposure resulted in 4 animals out of 40 that fully differentiate d as phenotypic female/genotypic male (ZZ)

adults. While the 4 animals cer tainly represent 10% of 40, th e total number of animals in the adult exposure segment of this study was 120. It is unc lear 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 adu lts, the data presented in the paper do not support such a global statement since the av ailable 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 effect s reported during the adult expo sure phase of the study is93 also uncertain. Finally, there ar e a number of uncertainties/d iscrepancies in the figures used to depict study results. Additional info rmation 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-revers ed ZZ frogs to produce the larvae used in the study. This information would speak to th e 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 b ack 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; howev er, it refers to the component elements of the rearing program as “treatments” and “replica tes”. 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 ho w measurement statisti cs relate back to

the overall number of animals used in each treatment or to the study as a whole. Additionally, animals that we re not part of the rear ing program depicted in Figure S1

were also utilized in this st udy even though they were not rear ed 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 gene ration would have to be carefully proofed using the genetic marker to ensure that the study used exclusively 100% ZZ males. According to the suppl ementary 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 stag e of the larvae at in itiation of exposure is not stated. The report simply states exposur e 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 constructi on material is not re ported. Adult rearing

tanks were constructed of fibe rglass 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 w hy 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 be low the chemical’s solubility limit of 33 mg/L (MRID 41379803) . Although the study reports the use of a solvent control, it does not repo rt 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); th erefore, 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-renewa l 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 Chow TM was used to feed larvae duri ng 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 we re “determined” as part of a morphometiric analysis. It’s unc lear, though, when these measurements were actually recorded and/or tested for significan ce. As discussed below, the morphometric analysis was confined to 9 control and 9 at razine-treated animals measured in December 2007 and a second set of 9 from each group sampled in December 2008.

Laryngeal weights (after fixa tion), laryngeal weight correct ed 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 reporte d as measured. Although no summary statistics are presente d on weight, the author s report that there was no statistical difference (p>0.05) in the size of the larynx, bu t that there was a statistical difference in

the structure.

No effects on size at or time to metamorphos is 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 us ed to monitor atrazine in both the ethanol

control and the atrazine treatm ent. Therefore, claims that the animals were exposed to atrazine for up to 3 years at 2.5 ug/L are unsubs tantiated in terms of the actual exposure95 level especially given the static renewal c onditions 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 st udies were conducted using 3 tanks with 40 animals/tank (120 animal s) 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 we re also used for the morphometric and histologic analyses. The study does not desc ribe 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 apportione d into 23-L tanks for experi ments involving fertility and

mate choice. It is uncl ear 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. Po st-metamorphic juvenile animals would then have been raised to adulthood during the fi rst 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-meta morphosis but rather 3 years post-hatch.

Figure 1 of the paper depicts a se ries of photographs of etha nol control and atrazinetreated males and stock females. Additionall y, some of the photographs depict atrazineexposed female (phenotypic female genotypic ma les). 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 pictur es is subjective wit hout evidence to indicat e that the scales are identical. Although th e authors attempt to emphasize differential development through the use of different-size brackets, th e 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 subjectiv e. More detailed image an alyses 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 fe males) 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 fe male genotypic males is uncertain.96 Figure 3 of the paper depicts representative larg est 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 de picts that breeding gland area (μm 2 ) 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-sectiona l area is minimally 220 x 180 μm or 39.6

mm 2 . As such, the breeding gland area in terms of μm 2 depicted on the y-axis does not appear to be consistent w ith the micrometer scale.

According to the study, the maximum cro ss-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-comparis on (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 tran sverse cross-sections depicted in Figures

3F

, 3G and 3H for ethanol control (ZZ) males, at razine-exposed (ZZ) males and stock (ZW) females do not demonstrate a marked di fference in the overall area for control and atrazine-treated males; however, the microgr aph depicting the atr azine-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 mor phological differences that coul d 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 overal l 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 th at “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 th is study is uncertain.

RT-PCR for cyp19 aromatase was conducted on onl y 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 includ ed in the analysis fo r 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 e ffects of atrazine on males to support the hypothesis that atrazine up-regulates aromatase. Additionally, the study incorporates animals (ZW fe males) that are not part of the study and as such do not reflect study conditions nor treatment with et hanol. In general, the number and scope of

animals assessed is not consistent wi th 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 assa y can be related to the overall atrazinetreated versus solven t 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 fe minized, 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 anim als 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, th is 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 cont ain sufficient information to determine whether mixed gender (phenotypic) existed; a lthough the method section reports that sex ratio was measured in a subsample of juvenile frogs (and then not report ed). It is unclear why this issue is not discussed and why resear chers have not been able to consistently replicate the previous mixed gender results.

Overall, there is uncertainty whether the continued reliance on hus bandry/environmental conditions during larval expos ure 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 ph ase of the study were likely

intended to minimize the extent of anesth esia required, suturing (marking the animals with thread) and cardiac punctu re are relatively invasive pr ocedures to perform without anesthesia and would likely cont ribute to stress levels among the study animals. As with other components of this study, the mate c hoice involved the use of animals (stock ZW females) that were not part of the study design.

Additionally, on the mating pair studies, the re port states that animals were allowed to mate overnight and that amplectant couples were identified the fo llowing 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. Howe ver, the available data [based on Figure 4A ] indicate that in each of the mating studies th ere was at least 1 fema le 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 a ffect breeding success and that

there was no difference in size between atr azine-exposed and control males; however, it goes on to say that in all of the trials, sm aller individuals from controls out-competed larger atrazine-exposed individuals.98 The text states that control males had signifi cantly higher plasma testosterone levels in the presence of females, when compared w ith 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 th e caption, solid horizon tal bars show mean

testosterone levels for successful males and open bars show the mean for unsuccessful

males. It is unclear from the informati on provided whether the statistical analysis

included both successful and unsuccessful male s 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 contro l 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 square s) and 4 atrazine-treated (red squa res) males. However, according to Figure S1 of the Supplemental Informati on, there is an N=9 in 2007 and an N=5 in 2008 provided that there was no ove rlap (points obscured by ot her points). Therefore, it

is unclear whether the lower N value is a re sult of data truncati on 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 ma y 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 major ity of [semeniferous] tubules lack mature sperm bundles and are nearly empty; however, Figure 5F that 6 out of 10 atrazi ne-treated animals had greater than 80% of th e tubules with sperm.

Although the authors cite a number of other stud ies in support of th eir current research, the authors’ interpretation of these other studies is not consistent with that of the EPA. Many of the studies cited have been review ed by both EPA science staff and the FIFRA SAP and these reviews differ substantially fr om 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 N ame: 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-nitrosodi ethanolamine 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 at razine, alone or in combination with sodium nitrite (NaNO2) as well as N-nitrosoatrazine (NAT) on amphibian larvae. Other chemicals were disc ussed 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 w ith 0.45 or 140 ppm NaNO2 at 20°C for 12 days. NAT (believed to be fo rmed 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 great er 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 conduc ted 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 incr ease in MN RBCs was si gnificant at 7.5 and 15 ppm but not 3.75 ppm.

Description of Use in Document: Qualitative.

Rationale for Use: While this study provides informa tion on the genotoxic potential of atrazine, there are uncertainties regarding th e test design and stat istical analysis.

Limitations of the Study:100 Limited water quality parameters were report ed. While the study indi cates that the study

was conducted at the maximum concentra tion 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 re ported 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 treatm ent, suggesting that one replicate was used per treatment and the results were from pse udoreplicates. If only one replicate was used, potential replicate vari ability 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 Liter ature 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 th e immune system of the frog, Rana pipiens ? Bios. 77 (4)

107-112.

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

To evaluate the report for potential affect s 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 at razine on the immune system of amphibians. It was hypothesi zed that exposure to increasi ng concentrations of atrazine

would correlate with a decr ease 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 chees ecloth covering opening. Jars kept at room temperature and solution wa s changed every 3 days and jars rearranged at random. Following standard protocols, sh eep red blood cells (SRBCs) were used as an antigen to induce immune response. After fr ogs were exposed for 4 or 8 weeks, SRBCs were injected intraperitoneally for five c onsecutive days. On the sixth day, the frogs were euthanized and spleens removed and ly mphocytes were recovered. Cells checked for viability and examined using a hemocytome ter. In trial 1, 9 of the 12 frogs were killed using 3 from each replicate and remain ing 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 fr og 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 infecti on based on an acquired immunodeficiency).102 For trial 1, there was a significant differe nce (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 (± st andard 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):

Invalid103 Rationale for Use:

Overall, there is a lack of reporting on certa in 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 so urce are not reported, th erefore, there is uncertainty in previous exposure of cont aminants to the frogs, water and food.

The preparation of the control and test concentr ations was not reported; therefore it is not

known if a solvent was used in the st udy. The study does not indicate if the

concentrations of the test solutions were m easured. Therefore, it is not known if the control represents a dilution water control or a solvent control.

Overall, there is some confusion by the revi ewer 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 Tria l 1, the reviewer is

unsure of how the animals were used as the study indicates “I n trial #1, nine of the twelve frogs were killed, using onl y 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 HP A, 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 a nd Figure 1 represent a sample size of 3 frogs per concentration which represents the nine of the twelve frog s previously discussed; if it represents results using potent ially different methods then there is uncertainty in the

results. The reviewer is unsure if in Trial 1 cell count s 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 tissu e 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 ther efore this observation confounds interpretation of any of the

results for this trial.

In addition, the study author indi cates 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 th at they reported that most of the frogs in trial 2 did not feed during treatment, interpreta tion of these results s hould 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 applicable105 3.22. Howe, G.E., R. Gillis, and R.C. Mowbray (1998).

Open Liter ature 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 LC 50 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 we re 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 a cclimation 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 minimu m of one exchange of water/hr. The larvae were acclimated to the test water for a period of 24 hrs prior to the in itiation of the test. At the onset of the test, earl y- 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 ch annel catfish were obt ained as fry from hatcheries; both were reared at the Upper Miss issippi 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 12 o 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 22 o 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 ca librated daily. During the study, mean water hardness was 44 mg/L as CaCO 3 , mean alkalinity wa s 33 mg/L as CaCO 3 , and mean pH was 7.9. The minimum oxygen concentrations exceeded 6 mg/L in all test chambers. Water temperature was maintained (± 1.0 o C) at 12 o C for rainbow trout and 22 o C for all other species.

Field-grade formulations of at razine 4L (40.8% a.i.) and alac hlor 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 indivi duals of each species (two replicates of 10 each) were exposed for 96 hr to each select ed 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. Th e concentrations remained within ±10% of the target concentrations throughout the study, except for tw o 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 LC 50 s.

Results:107 The study resulted in the following 96-hr LC 50 s (see Table 1 ):

TABLE 1. 96-hr LC 50 Values for Alachlor, Atrazine and Atrazine + Alachlor for the Leopard Frog, American Toad, Rai nbow Trout, and Channel Catfish.

SPECIES (STAGE) LC 50 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 amphi bians appear more sensitive than fish to the chemicals. Additionally, the toxicity of the 50:50 mixture of atrazi ne + alachlor appeared significantly greater than additive at 96-hr of exposure for all test species and stages except the rainbow trout, indi cating 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 th e onset of metamorphosis. Because of this, the authors st ate that acute toxicity data presented only for early-stage larvae should be interpreted with caution be cause 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 iner t 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 applicable109 3.23. Kerby, J.L and A. Storfer. 2009

Open Liter ature 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 chlo rpyrifos on susceptibility of the tiger salamander to Ambystoma tigrinum virus. Eco. Heal. . (ECOTOX Ref No. 121497)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 at razine 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 technica l grade atrazine [0, 20, 200 μg /L]; 4 concentrations of chlorpyrifos [0, 2, 20, 200 μg/L]; and eith er the ATV or not. Control treatments contained methanol solvent [reviewer calcula ted (1000 μL of 10% methanol solution in 500mL = 0.2 mL/L)]. Larvae were reared in th e 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 replicat e. Some replicates

received the Ambystoma tigrinum virus (virus conc. at estimat ed LC50) via water bath for the first7 days of the test. Test soluti ons 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. Vi rus loads and infection status of larvae were analyzed using real-time PCR using tail tis sue and measured in triplicate. All virustreated animals examined along w ith random sample of no-virus controls to verify lack of

contamination.

Results

According to the authors, there was no signifi cant main effect on mortality for atrazine, or interactive effects of atrazine by chlropyr ifos on mortality. However, there was a significant interactive effect s of atrazine by virus ( p = 0.03) with atrazine slightly110 reducing mortality in no-virus treatments and slightly increasing mortality in virus treatments (Figure 2).

Figure 2. Proportion of salamander larvae

that survived viral a nd 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 atra zine [data not shown].

Figure 3. Proportion of salamander larvae th at survived viral and pesticide exposur e. Virus only data presented to show additive effects of increasing pesticide concentrations. Logi stic 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 a bout survival of the tiger salamander from atrazine exposure as well as the possible in teractive effects of atrazine and a virus on111 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 no t 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, ba sed 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 t ype 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 surviv ing 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 fo r 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 confid ence intervals around that LC50. Therefore, while it does seem that there is a slight increase in mo rtality 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. Ba sed 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 applicable112 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 trem atode 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 la boratory studies with three chemicals (malathion, esfenvalerate and atrazine). Th is review will focus on the laboratory study with atrazine. Wood frog embryos (Rana sylv atica) embryos (collected from three ponds in Centre County PA (two from each pond)) a nd 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 place d individually in polyethylene tubs in

3.5L test solution at atrazine concentrati ons 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 so lvent control (% solvent in test solutions not provided) were used. Tadpoles kept at 16:8 light:d ark cycle for 4 weeks and solutions changed every other day. After 4 weeks, tadpoles pl aced 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 tadpol es for leukocytes enumeration, and then the tadpoles were euthanized and preserve d for metacercarial cysts enumeration.

The authors focused on the response of eosinophi ls because of the role they play in macroparasite infections. They state that e xposure 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 propor tion 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).113114 Figure 1. Summary of effects of pes ticides 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 solven t 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 a nd 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 re ported. 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 c ontainers 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 use d. 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, ther e is uncertainty in exactly what treatment groups ar e statistically significant.

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, Office of Pesticide Programs115 3.25. Kloas, W., I. Lutz, R. Urbatzka, T. Spr inger, H. Hrueger, J. Wolf, L. Holden, and A. Hosmer. 2009 Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Numb er 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 th e potential transient impacts of atrazine on sexual differentiation pro cesses in Xenopus laevis.

Twenty-five sexually undifferentiated (sta ge 48) and differentiated (stage 58) Xenopus

laevis tadpoles exposed to 25 μg/L atrazine a nd a control. Each treatment had two replicates as described previ ously in (Urbatzka 2 007) and test solutions were replaced every 24 hours. After 4 days, 4 undifferen tiated tadpoles were randomly sampled per tank (n=8). For the different iated tadpoles, after 4 days exposure, they were randomly sampled from each tank until 4 males and 4 fema les 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 anal ysis conducted on entire complex. Kidney and gonads were separated for sexually differe ntiated tadpoles. According to the study authors, in the testes, aromatase expression wa s 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 br ain or kidney-gonadal complexes compared to

control.

Description of Use in Document (QUAL, QUAN, INV):

Qualitative116 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 c onstitutes the control are not specified. Water quality parameters, measur ement of atrazine in test solutions, and survival not specified. As only two tanks we re 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 aromat ase expression in the testes and high expression in the ovaries. As there was aromatase detected in all the kidney-gonadal complexes in the undifferen tiated tadpoles, the st udy authors state the

renal tissue contains aromat ase. However, while the gonads were not wholly differentiated, the fact that the kidney-gonadal complexes di d contain gonadal tissue and

that aromatase was detected in the ovarie s, and kidneys were not measured in the differentiated tadpoles to confirm aromat ase expression, may it be possible that aromatase was present in the gonada l 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 applicable117 3.26. Kloas, W., I. Lutz, R. Urbatzka, T. Spr inger, H. Hrueger, J. Wolf, L. Holden, and A. Hosmer. 2009 Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Numb er and Citation: Kloas, W., I. Lutz, R. Urbatzka, T. Springer, H. Hrueger, J. Wolf, L. Holden, and A. Ho smer. 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating the potential eff ects 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 fo rth 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. 2007118 3.28. Koprivnikar, J., M. R. Forbes, and R. L. Baker. 2007

Open Liter ature 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-para site interactions: atrazine, frogs, and

trematodes. Environ. Toxicol. Chem. 26 (10): 2166-2170.

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 we re behaviorally mediat ed. 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 we re obtained from egg masses that were collected from pond near Ca sselman ON, Canada; the authors stated site not tested for contaminants but pond receiv es no agricultural runo ff. Tadpoles were individually exposed in 4L plastic tubs that contained 1.8L test so lution that was renewed every 3 days and were mainta ined at 21oC for 31-days. Ce rcariae were obtained from snails that were field collected. For the expe riment, tadpoles were pl aced into containers containing 50mL dechlorinated water and were exposed to 8 cercariae that were not exposed to atrazine. For experiment where cercariae exposed to at razine, the cercariae were exposed to corresponding atrazine con centrations for 1 hour before being placed into container with tadpole. 6 tadpoles we re used for each treatment group. For five of the six tadpoles exposed to atrazine but cont rol cercariae, behavior observations were made by videotaping the organisms and record ing 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, tadpo les 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 gr oups and control cercariae. There was a119 significant difference in cyst abundance and intensity between cont rol and 30 μg/L group exposed to control cercariae.

The authors indicate that th e presence of control cercari ae 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 addi tion, replicate variabil ity was not reported and effects were only observed when tadpoles , not cercariae were exposed to atrazine therefore, there is uncertainty in environmen tal 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 applicable120 3.29. Koprivnikar, J. 2010

Open Liter ature 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 (cercari ae) exposure (yes or no) for a total of 12 treatment group (excluding th e negative and ethanol solvent control (100 μg/L)).

Tadpoles were obtained as eggs from Ca rolina Biological Supply Company (Burlington, NC) (six batches of eggs r eceived and unknown if from sa me clutch) and raised in declorinated tap water and fed boiled spinachand rabbit chow. When tadpoles reached Gosner stage 25-26, they were individually tr ansferred 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 th e cups contained cercariae obta ined from naturally infected

P. trivolvis collected from Santa Clar a County CA. After cerca riae exposure, tadpoles were individually transferred to a 2-L plasti c 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 (r eport stated previous work

indicated minor loss of atrazi ne 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 we re placed into containers to allow for predation by dragonflies. The dragonfly larvae were rotated through the121 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 we re 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.

F IG . 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 ra nged from 95 – 100% and was not analyzed for interactions (Figure 1). Not all tadpoles exposed to cercariae b ecame infected (n=26) and so data was analyzed by including and excluding the uninfected tadpoles. According to the study author, a signif icant difference among the three test concentrations was observed for survival; th e 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 mort ality was lower in parasite-e xposed tadpoles but higher in tadpoles exposed to 3 μg/L atrazine; but th at 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 (Figur e 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 un infected tadpoles negatively impacted developmental stage and that expos ure to 3 μg/L atrazine decreased both mass and development stage. Exposure to 3 μg/L atrazine had a significant overall effect by122 decreasing both mass (p < 0.0001) and devel opmental stage (p< 0.0001) when parasite exposed but uninfected tadpoles were included or excluded. The author states that the significant interaction between parasite in fection and exposure to 3 μg/L atrazine

occurred because the mass of in fected 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 pr edator 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 nega tively impacted development stage for both.

F IG . 2. Tadpole mortality due to (a) parasite and predator cue (larval Anax dragonflies) exposure, and (b) parasite and 3μg/L atrazine exposure. Nota tions of – and+ indicate absence and presence, respective ly, of exposure to factor.

The study author states that exposure to pa rasitism had little overa ll effect on larval amphibians in this study, however it is clear th at 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-in fected(+) and uninfected (-) t adpoles 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 at razine (dot-dashed line) or 0 μg/L atrazine (solid line). Differ entiation 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 eff ects (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 reported124 As this study was conducted wi th two atrazine concentrations and one of them resulted in ca. 100% mortality, the reviewer could not de termine 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 discer n 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 na ked eye (no obvious hind limb bud), therefore there could be some uncer tainty 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 reporte d 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 au thor 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 whet her 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 a ppears to better correlat e with the % mortality values reported in Appendix A as, for exampl e, presence of both pr edator and parasite result in ca. 25% mortality which is the exac t value reported in th e Appendix. However, there is uncertainty in what the back corner bar represents because it does not appear to correlate with values in Appendix A. Therefor e, 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 whic h the absence of atrazi ne 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 indicat ion of significant effects for wh ich treatments in table form for example would have been helpful.

Based on the values reported in Appendix A, th ere appears to be some variability in the mortality response for the tadpoles. Accordi ng to Appendix A, mortality in the pooled controls was 16%. While the mortality of tadpol es exposed only to 3 μg/L atrazine was 31%, tadpoles exposed to both atrazine and pred ator 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 cu es reduced mortality which is not the expected response. In addition, the mo rtality 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 pa rasites and to all th ree stressors had 25% and 44% mortality, respectively, given that ta dpoles exposed only to predator cue had125 31% mortality and the variability in mortal ity between atrazine exposure and predator cues, it is difficult to conclusively determ ine that atrazine negatively impacted tadpole mass and developmental stage. Also, if mortalit y was significantly impacted at 3 μg/L as it appears to be based on th e discussion, it may have influe nced evaluation of the other endpoints given that the frogs may have expended energy on de toxification rather than on growth and development. However, the study author states the smaller tadpoles were likely not selected against due to the mortalit y 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 applicable126 3.30. LaFiandra, E.M, K.J. Babbitt, and S.A. Sower. 2008

Open Liter ature 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. Toxi col. Environ. Health, Part A. 71: 505-511. (ECOTOX 103336)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 th e effects of a nonlethal predator and exposure to atrazine and their interacti on 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% relate d compounds) and exposure or not to a nonlethal predator, dragonfly larva (A nax sp) which was placed in a mesh cage in aquaria with tadpoles; tadpoles not exposed to nonlethal pr edator contained an empty cage. Tadpoles were obtained from a pair of adults collected while in amplexus (location not specified). Three tadpoles we re 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. Followi ng 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 remove d 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 no t 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 signi ficantly reduced the proportion of tadpoles that initiated metamorphosis at 200 ppb. Surv ival 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 predat or 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 atra zine contained ovaries consisting of nearly equal proportions of oogonia and previtellogeni c 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. Howe ver, 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 developmen t 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 laborat ory. While loading was within recommended limit, limited reported water quality with re latively long renewal period(50% renewal every week) may have potential environmen tal condition issues. Degradates were

measured in atrazine soluti ons and since control solutions were not reported there is128 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 Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable129 3.31. Langerveld, A.J., R. Celestine, R. Zaya, D. Mihalko, and C.F. Ide.

2009.

Open Liter ature 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 expos ure to high levels of atrazine alters expression of genes that regulate immune a nd growth –related function in developing Xenopus laevis tadpoles. Environ. Res. 109: 379-389. (ECOTOX Ref No. 121497)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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-fert ilization), 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 pr epared 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 st udy authors, the original test design was to collect tadpoles at stage 66 to compare to other studies, how ever, 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 ev aluated for gender, gross morphological abnormalities. Endpoints measured for surviv ing stage 62 tadpoles include: weight, snout-to-tail length, and gross morphological (g ender, fat bodies) or molecular analysis (gene expression); in tadpoles used for ge ne 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 Affymetr ix GeneChip® protocols. Five genes were chosen from the final Affymetrix datase t for validation using Taqman qRT-PCR: pancreatic trypsin, chitina sse, chitobiase, neurohormone- like peptide and magainin II copy E.130 Results

Survival in the 400 μg/L treatment group wa s 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 analy zed by gender. Male and female tadpoles exposed to atrazine had a significant reducti on 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 me tabolism, 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 th e analysis showed significant increase in

chitinase.

Description of Use in Document (QUAL, QUAN, INV):

QUAL for survival; INVALID fo r other endpoints due to significant mortality observed at only atrazine con centration tested.

Rationale for Use:

This study provides information on survival for Xenopus laevis tadpoles at a single concentration. However, as some environm ental 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 chr onic 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 fo r 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 vesse l which contained 1 liter solution. Based on the graph in Figure B, the m ean body weight of stage 62 ta dpoles appears to range from ca. 0.35 – 0.48 g [reviewer visually determined this range from figur e]; if there were 4 tadpoles/vessel then the mass/ vessel would exceed the recomm ended 1 g or 1 tadpole/L.

Environmental conditions other than temp erature 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 numbe r that were exposed is unclear. The study131 indicates 15 dishes for the at razine group and 20 dishes for the control were set-up and

that each dish contained no more than 4 ta dpoles, 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 th e control and atrazine group, respectively (reviewer presumed that not all dishes contai ned 4 tadpoles and that this was the initial number of exposed animals). The study stat es 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 ex cluded and those include d 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 we re all reported to be for st age 62 tadpoles and therefore do not appear to take into account any deaths from tadpoles entering st age 63 or higher as previously noted. Therefore, it appears that there must be some mo rtality in the excluded tadpoles to account for the statement that tadpoles entering 63 or higher were dying, but the number of mortalities are not reporte d. The reviewer assumes that the overall mortality in the control is w ithin acceptable limits.

As survival was affected in the 400 μg/L atr azine group, it is not wholly unexpected that other sublethal effects such as growth and ge ne 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 precl udes 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 applicable132 3.32. Langlois, V.S., A.C. Carew, B.D. Pauli, M.G. Wade, G.M. Cooke, V.L.

Trudeau. 2010 Open Liter ature 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 herb icide 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 metamorphos is in the Northern leopard frog. The hypothesis was that exposure to atra zine alters estrogen receptor ( α ) (eralpha) mRNA

level and 5 β -reductase (srd5beta) activity in the target ti ssues of exposed tadpoles.

Methods

Northern leopard frogs (Rana pipiens) we re 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) a nd the kidney-gonad complex was removed and fixed for histology. Atrazi ne (n=2/site per date) was measured from five different

locations in the reference site ever y 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 referenc e site and reared in laboratory at density of 1 g tadpole/L) were added to each mi crocosm at density of 0.1 g tadpole/L. Mesocosms (aged 378-L high-density polyeth ylene tank) contai ned 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 formul ation 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 12 th and 19 th ). Dissolved oxygen, pH, and temperature recorded weekly; when tested, ammonia leve ls below detection (250 μg/L). Atrazine

concentrations in mesocosms measured when spiked with atrazine and then roughly133 every 15 days [reviewer based on figure provi ded 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, a nd snout-to-vent lengt h (SVL). Sex was visually determined, and brain, liver, and ta il removed and samples frozen; kidney-gonad complex fixed. Subset of male samples histol ogically analyzed for presence of testicular oocytes (serially sectioned longitudinally; b lind analysis). At end of experiment, premetamorphic G34 tadpoles were analyzed fo r 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 ( dio 2 and dio 3, respectively, and ribosomal protein L8 ( rpl8 ), and neuropeptide corticot rophin-releasing hormone ( crh ); samples were conducted in duplicate. Enzymatic ac tivity of cyp19 and srd5beta wa s 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 gr oups, 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 stat e that possibly contaminated ground water used to keep mesocosm water levels up accounted for detects in contro l. No statistical difference in pH, temperature or dissolve d 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, signi ficantly 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 SV L 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 fo r 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) 1:0.6134 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 cyp 19mRNA level or activity in the brain of the G42 animals but EE2 did; also no changes in c yp19mRNA levels in brains of G34 tadpoles. In the brains of the G34 tadpoles, expre ssion of eralpha in the 1.8 μg/L group was 2.5 times greater than control, but no difference in the brains of G42 ta dpoles or livers from G34 and 42 tadpoles or in EE2 group for any tis sue or stage. Ther e 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 su rvival and metamorphic success of the northern leopard frog, and 5 mesocosms per treat ment allow for greate r 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 fr om a reference site that was surrounded by streams in which atrazine was detected (ra nge 0.01-1.6 μg/L); ther efore there is some uncertainty regarding previous exposure to atra zine and other potential contaminants. In addition, several sources of food and water were provided to animals and the report made no statements about possible cont aminants 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 bu t do not report the percentage of atrazine in the formulation [reference on Sygenta Canada website indicates it is 43.7%, http://www.syngentafarm.ca/prodrende r/index.aspx? nav=labels&ProdID=778 ]

The water control treatment group had measur able 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 ta dpoles were placed into the mesocosms at stage 25 and exposure to the chemicals starte d when tadpoles were at stage 27. The study did not report how observations of the frogs was conducted to determine when at stage 27135 as well as how observed during the expos ure and if survival/mortality was only

determined at test termination.

Survival and metamorphic success were repo rted based on total number of organisms exposed and not as a replicate mean with e rror bars reported; th erefore, variability between replicates is not known. While no significant differences between groups during the study, there was some noticeable variati on 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 at razine group. Also, based on Supplemental figure 3, it appears that th e dissolved oxygen dropped be low 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 fema les in the controls and the reference site. As there was mortality in control and treatm ent groups (21% in water control and 25 and 34% in the 0.1 and 1.8μg/L atrazine groups, resp ectively) 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 presenti ng sex ratio data on frogs at G42 was not stated in the study. The study st ates that animals were rem oved and processed when they reach G42, therefore it seems that every frog that that underwent complete metamorphosis should have been able to be se xed. 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 subs et of animals that completed metamorphosis there is uncertainty in how this affected the results.

The study authors state that this study pr ovides evidence that atrazine may feminize R. pipiens when exposed to low levels of atrazine and that they sugge st that associated changes in brain eralpha mRNA level and liver srd5beta activity might be involved in producing the response. Now while the con centration 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 th at 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 author s state that there is data lacking in the importance of this dimorphism, the sex rati o 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 indi cate 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 inform ation that atrazine alte rs 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/L136 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 applicable137 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 re view 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 expo sed 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) plasmacorticosterone and thyroxine concentrations, (2) larval size, a nd (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 tr eatment compared w/ both the control and the 250 ppb treatment group. Larvae exposed to 75 ppb atrazine reach ed stage 4 later, but at a size and weight co mparable to the control group. Larvae in the 250 ppb treatment progressed to stage 4 at the same tim e, but at a smaller si ze 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 e ffect of elevated thyroxine by slowing metamorphic progress and allowing additional gr owth between stages. The advantage is transformation to a larger size, with higher survival and fecundity prospects. Larvae subjected to 75 ppb atrazine t ook longer to undergo metamor phosis, indicating that they may spend more time in the wetland before me tamorphosis (failure to transform before cold temps set in, thus making them vulnera ble to anoxia in ice-covered wetlands). Larvae subjected to 250 ppb atrazine comp leted transformation at a smaller size (indicating potential lower survival and fecundity).

Stress hormones (e.g., corticosterone) and th yroid hormones (e.g., thyroxine) promote

differentiation associated with the onset of metamorphic climax. Plasma corticosterone138 and thyroxine concentrations ri se in response to stressors in the environment as well as during metamorphoric climax. Corticostero ne 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 la rvae that have not reach ed 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 we re 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 i ndividually 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 CaCO 3 ).

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 temper ature 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 Programs139 3.34. Lenkowski, J.R., J. M. Reed, L. Deininger, and K. A. McLaughlin.

2008.

Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Numb er 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 Pers pect. 116 (2): 223-230

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 at razine exposure during organ morphogenesis in Xenopus laevis . 2,4 D and glyphosate were al so 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 atrazi ne 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 st age resulted in 100% mortality. A trial using both DMSO and

ethanol was conducted to determine which so lvent 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 lo wer toxicity than ethanol. DMSO solvent concentrations ranged from 0.1 to 0.35%. Solutions were renewe d every 24 hours and organisms monitored for mortality and malforma tions. At test termination, tadpoles were scored for circulatory malformations an d then fixed and scored for edema and malformations of the heart, intestine and axis. Antibody localizat ion, apoptosis in the brain and kidney was also evaluated. All e xposures were performed two or three times with different tadpole broods.

Result s

Mortalities were <5% in treatments and di d not differ among groups . The study authors

stated that malformations of the heart a nd visceral hemorrhaging and that atrazine significantly increased heart malformations acr oss 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 certa in 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 th e membranes. The authors evaluated DMSO

and ethanol for observed toxic ity. While the incidence of observed toxicity was higher with ethanol, when combined with atrazine , the percent inciden ces using DMSO were always greater, unless 100% effect was obs erved in both solvents , with DMSO than ethanol suggesting that DMSO enhanced toxi city. 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 applicable141 3.35. Lenkowski, J.R. and K. A. McLaughlin. 2010.

Open Liter ature 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 a nd retinoin signaling during organ morphogenesis in Xenopus laevis

. J. App. Toxicol . 30: 582-589 (ECOTOX Ref No. 153851)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 characteri ze tissue and molecula r level effects of atrazine exposure durin g 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 modi fied Ringer’s (MMR) solution and embryos staged according to Nieuwkipp and Faber (1994). I n results section study authors state that NF stage 41 was just prior to beginning experimental exposures. Atrazine stocks were prepared in dimethyl sulfoxide (D MSO) and diluted in 0.1xMMR. Final DMSO

concentration was 0.35 %. Test solutions we re 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 imm unohistochemistry (IHC) was employed and muscle structure was examin ed 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 ha s described roles during X. laevis development (MMP2, MMP9TH, MMP11, MMP13, MMP18); three biol ogical replicates were used. Zymography was used with 20 pooled tadpoles per treatment and protein concentrations were measured using the Bio-Rad Protein Assa y and were repeated wi th four biological142 replicates. For the tadpol e intestines, hybridization ( in situ ) was used and hybridization was visualized and representativ e 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 st ate that there was a si gnificant effect of

treatment across all MMPs examined with MMP9TH and MMP18 both increased expression with dose but only MMP18 signifi cant at 3.5, 25 and 35 mg/L (Figure below). A RT-PCR analysis in a time cour se 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 signi ficant at 6 and 48 hours for 35 mg/L. Zymography used to determine if atrazine altered MMP enzyme ac tivity, and at 10 and 35 mg/L, activity was decreased and increased comp ared to control; authors stated that is probably corresponded to MMP13. In the intest ines, after 48 hours exposure at 10 and 35 mg/L, gene (Xcyp26) expression signifi cantly decreased and gene XRALDH2 also significantly decreased at 35 mg/L. The study au thors 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 thr ough modulation of MMPs and the disruption of retinoic acid signaling. They al so state that the data suggest that acute atrazine exposure causes the up-regulation, or pr events down-regulation, of spec ific MMPs. In addition, they state that acute atrazine exposure with changes in MMP transcript le vels 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 obser ved down-regulation of intestinal fatty acid binding protein in the intestines which they state is in res ponse 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 contro l 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 at razine-treated tadpoles are indicatedwith white arrows. Significance was determined by two-way ANO VA 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 solv ent as DMSO can promote

movement across membranes and the solvent co ncentrations used were greater than the U.S. EPA guideline recommended concentrations.144 Limitations of Study:

Test vessels used, loading, a nd 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 us ed in this study (0.35 %) is 7 or 35 times greater than the

recommended solvent concentration for U.S. EPA OPPTS acute stat ic (0.05%) or acute flow-through and chronic testing (0.01%), respectively. All so lvent concentrations tested (0.035 to 0.35% DMSO) are greater than the recommended 0.01%. It is also not entirely clear why three different solven t concentrations were used and how they relate to the

atrazine concentrations.

The authors state that acute exposure to atr azine disrupted the stru ctural integrity of differentiated cartilage and musc le and distorted cranial cartil age 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 Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable145 3.36. Lenkowski, J.R, G.Sanchez-Bravo, K.A. McLaughlin. 2010.

Open Liter ature 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 ex posures have diverse effects on Xenopus laevis organ morphogenesis. J. Environ. Sci. 22(9): 1305-1308. ECOTOX Ref No. 153876)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 atra zine (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 negativ e 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 replicat es 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 fixe d, prepared and incubated overnight with a primary antibody known to identify X. laevis muscle sarcoplasmic reticulum. Antibody

localization was visualized w ith a chromogenic reaction.

Results There was no mortality in the atrazine expos ures. Intestinal malf ormations and edemas were significantly increased in the 3.5, 10 a nd 35 mg/L atrazine concentrations. Also, according to the study authors, the cranial musc les 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 so lvent as it can promote movement across

membranes.

Limitations of Study:

DMSO was used to prepare the atrazine con centrations. Solvent control concentrations of 0.1and 0.35% DMSO were reported as test co ncentrations. DMSO is not a preferred solvent as it can promote movement across me mbranes 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 wa s 2 and 7 times that recommended level. While not explicit in the study, the reviewer as sumes that the solvent concentration in the atrazine treatment groups wa s either 0.1 or 0.35% DMSO.

Environmental conditions other than temp erature were not reported and although no mortality was reported, the reviewer cannot determine whether the tadpoles may have

been stressed due to other environmental fact ors. Test vessels we re not described.

Loading in terms of mass tadpole/volume so lution was not reported but rather in number of tadpoles/10mL. The use of 13-18 tadpol es/10mL solution may have increased the loading beyond the re commended 1 tadpole/L.

While Figure 1 states that the DMSO controls were not significantl y different than the non-treated controls, it is then presumed that the control refe renced in Figure 1 is for the non-treated control given that th e number of organisms is si milar to the treatment groups and does not appear to repr esent 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 Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

PC Code: 080803

ECOTOX Record Numb er 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 Pers pect. 116 (2): 223-230

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 at razine exposure during organ morphogenesis in Xenopus laevis . 2,4 D and glyphosate were al so 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 atrazi ne 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 st age resulted in 100% mortality. A trial using both DMSO and

ethanol was conducted to determine which so lvent 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 lo wer toxicity than ethanol. DMSO solvent concentrations ranged from 0.1 to 0.35%. Solutions were renewe d every 24 hours and organisms monitored for mortality and malforma tions. At test termination, tadpoles were scored for circulatory malformations an d then fixed and scored for edema and malformations of the heart, intestine and axis. Antibody localizat ion, apoptosis in the brain and kidney was also evaluated. All e xposures were performed two or three times with different tadpole broods.

Result s

Mortalities were <5% in treatments and di d not differ among groups . The study authors

stated that malformations of the heart a nd visceral hemorrhaging and that atrazine significantly increased heart malformations acr oss 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 certa in 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 th e membranes. The authors evaluated DMSO

and ethanol for observed toxic ity. While the incidence of observed toxicity was higher with ethanol, when combined with atrazine , the percent inciden ces using DMSO were always greater, unless 100% effect was obs erved in both solvents , with DMSO than ethanol suggesting that DMSO enhanced toxi city. 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 applicable149 3.38. Oka, T., O. Tooi, N. Mitsui, M. Mi yahara, Y. Ohnishi, M. Takase, A.

Kashiw agi, 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. Shin kai, N. Santo, T. Iguchi. 2008 Effect of atrzine on metamorphosis and sexual differentiation in Xenopus laevis

. Aquat. Toxicol

. 87: 215-226. (ECO TOX Ref No. 103345)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 a nd 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 atrazi ne, 0.27 μg/L E2, and 0.01% ethanol control; one replicate tank (30 tadpoles) was used fo r each treatment and frogs collected at metamorphosis. Stage 66 frogs were sexe d 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 pr imary-cultured hepatocytes wa s also conducted but is not

discussed in this review as it is an in vitro assay.150 Results

Measured atrazine concentra tions 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 concentra tions 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 repo rted for one of the two tanks in the 0.1 and 100 μg/L, 73.3 and 26.7%, respectively; as su ch, these two tanks were not used in subsequent analyses. Sex ra tio was significantly different for the 10 and 100 μg/L atrazine groups compared to the contro l; no hermaphroditism was observed (Table 1). According to the study authors, evaluation of sex by gross morphology was corroborated by histological analysis. Time to meta morphosis was similar among treatment groups and there did not appear to be an accelerati on or inhibition of development [statistical results/p value not reported].

In experiment II, survival was >96%. Accord ing to the authors, th ere 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 endpoints151 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 contro l as well as the variability in survival

for two of the atrazine treatments and the m easurement 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 co ntrol. Therefore, potential effects from

the solvent could not be evaluated.

The food source for the adult frogs used as th e source of eggs for the study was reported to show slight estrogenic activity in the y east 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 th e 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 th e two replicate tanks for the 0.1 and 100 μg/L groups, respectively. A source of this variab ility 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 me tamorphosis not reported.

Total number of animals with ovaries, testes, and hermaphroditism and survival per treatment group are reported. Variabili ty between replicat es not reported.

The reported measured concentr ations 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 con centrations 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 applicable152 3.39. Olivier, H. M. and R. R. Moon. 2010.

Open Liter ature 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 salamande r embryos and their symbiotic alga. Ecotoxicology 19: 654 – 661. (ECOTOX Ref No. 153629)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 af ter laying) of spotted salamanders ( Ambystoma

maculatum ) collected from pond in Evangeline Pa rish, Louisiana. Each egg mass, consisting of 26 - 127 eggs, constituted th e treatment unit; 20 egg masses assigned to each treatment group consisting of the atrazi ne 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 renewe d on exposure day 21. Individual egg masses were enumerated (number of eggs per egg ma ss) and were maintained in deionized water within 0.75L plastic contai ners. 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 record ed. At the start of the study, Harrison development stages ranged between 10 – 17. Algal growth determined by scoring the intensity of green coloration agai nst 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. Ha tching success for each egg mass determined by dividing the number of hatchlings by the numb er 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 co mplete bleaching which was interpreted by the study authors as complete mortality of the al gae and “probably led to severely reduced oxygen production.” Control egg masses displa yed 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 da ys in all but one egg mass. Timing and mortality of egg masses are reported to be di rectly related to atrazine concentration.

Based on the figures provided, the study app ears 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 si gnificantly (p<0.05) inversely related to atrazine concentra tion. 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 atra zine exposure affected the spo tted 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 provid e any water quality information and a number of assumptions are made regarding the c ondition of algae and potential photosynthesis/oxygen depletion. While effects on [apparent] chlorophyll and mortality are documented, the use of a formulat ed 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 ingredie nt; 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 produc t 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 masse s 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 be tween 10 – 17 suggests that the embryos were not in synchronous development.

Use of deionized water; authors claim that a re trospective analysis of the results indicated

that the use of deionized water did not im pair the successful hatching, development and

larval survival of controls. Deionized wate r was selected since “ it would not harm the eggs or larvae and because it would not in troduce 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 3 o C to 10 o C.

Authors relied on an aggregate color assessm ent to determine status of algae; they equated the visual absence of color to algae death [and oxygen depletion] and did not verify whether this assump tion was justified and whethe r oxygen concentrations had actually been depleted.

Authors acknowledge that th e use of formulated endpr oduct prevented them from assigning any effects observed in th e study exclusively to atrazine.

The authors acknowledge that it was difficu lt to accurately determine the number of embryos inside the large three dimensional egg masses and therefore there were “some” discrepancies between the in itial count and final counts /unhatched eggs and they excluded from analysis those egg masse s for which discrepancies were >10%.

It is unclear from the study results whether mo rtality of the egg masses refers to the entire egg mass or individual eggs within the egg ma ss. It is note worthy that the reported “pathologies” were initiated immediately after the failure of the climate control units, that the pathologies were transito ry (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 A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable156 3.40. Orton F., I. Lutz, W. Kloas, and E.J. Routledge. 2009 Open Liter ature 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 (recombinan t 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 atrazi ne 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 applicable157 3.41. Relyea, R. A. 2009

Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Relyea, R. A. 2009. A cocktail of contaminan ts: how mixtures of pesticides at low concentrations affect aquatic commu nities. 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 rega rding effects of atr azine 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, car baryl, chlorpyrifos, diazinon, and endosulfan) and five herbicid es (glyphosate, atrazine, acetochlor, metolachlor and 2,4-D) at low concentra tions on outdoor mesocosms composed of zooplankton, phytoplankton, periphyton, and larval amphibians (gray tree frogs, Hyla

versicolor and leopard frogs, Rana pipiens ). The pesticides we re 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 meso cosms 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 consis ted of only 3 replicates). The treatments158 were as follows: a negative control (water), a vehicle control (ethanol ), each of the five insecticides applied separa tely (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 tank s were filled (on May 15- 19, 2006) with about 1000 L of well water (pH 8). On June 7 th , 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 16 th , 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 25 th ), the tadpoles were added. The tadpoles were collected for the experiment as newly oviposited egg masses and hatched in 200-L wading pools where th ey 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 31 st ; 23 masses of gray tree frogs, collected on May

14 th and 17 th ), thus the two species were of differen t 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 (ini tial mass ± SE: gray tree frogs, 77 ± 4 mg; leopard frog, 134 ± 12 mg). The density of tadpoles in the experimental tanks was 9/m 2
for each species, which was within natu ral 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.) us ing ethanol as the vehicle. Ethanol

concentration was 0.003% in the solvent contro l tanks and the tanks receiving a mixture of all ten pesticides (ethanol concentration in the atrazine-onl y treatment was not stated).

Control tanks without ethanol were also incl uded. Atrazine was added to the tanks and

the surface water was mixed with a 500-mL co ntainer. 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. Th e measured concentration of atrazine was 6.4 μg/L (p.p.b.). The control tanks were not an alyzed 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 an alyzed for any of the pesticides.

The temperature, pH, and dissolved oxygen in each tank were measur ed 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 cen ter of the tank using a 0.2-L tube sampler. The five samples from each tank were combined, f iltered (62-μm Nitex screen), and then preserved in 70% ethanol. The zooplankton wa s classified to species. Twelve taxonomic159 groups of cladocerans and copepods were de tected. 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 vi a 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 sample d via scrubbing one side of the unglazed clay tile into a tub of filtered water. Afte r 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 biom ass of the periphyton.

Metamorphs were removed from the tanks when they had at least one emerged forelimb (stage 42); the gray tree frogs emerged firs t due to their inherently shorter time to metamorphosis. Daily searches of each ta nk were conducted starting day 21 (July 17) of the experiment when the first metamorph was observed through day 57 (August 22 nd , last day of experiment). After removal, the metamo rphs 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 e xperiment to stage 46 was defined as time to metamorphosis. Each animal was euthani zed in 2% MS-222, pres erved, 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 or der 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 us ing multivariate ANOVA (MANOVA). The MANOVA was composed of the second meas urements 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 meta morphosis 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 MANO VA. Each univariate response variable was examined using eith er an ANOVA (for responses that were measured at the end of the experiment: tr ee frog mass, tree frog time to metamorphosis,160 tree frog survival and leopard frog surviv al) or a repeated-measures ANOVA ( for responses measured twice during the experime nt: temperature, disso lved 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 necessar y. 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

, F 182,313 = 2.5, P < 0.001); therefore, the ANOVA analyses were con duct 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) rega rding 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 te st) 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 sign ificantly 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) rela tive 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 mixt ure 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 tr eatment 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). Ph ytoplankton 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 se parate insecticides, five separate herbicides, and a mixture of all ten pesticides. Data are mean ± 1 SE on a log scale. Wi thin 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, se e Figure 1. Figure 4 from Relyea 2009.

Abiotic Effects

Tank water pH and dissolved oxygen were meas ured 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 ox ygen over time in outdoor mesocosms exposed to no pesticides (control), solvent only (ethanol), five se parate 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 Fi gure 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 th e biomass of any of the trophic groups monitored in this outdoor mesocosm e xperiment. The only significant effect of

atrazine treatment which wa s observed was the increase in mass at metamorphosis for gray tree frog tadpoles ( P

= 0.045). The study author repo rted that the mechanism for

this effect was unclear. The overall effect s 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) an d (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 al gae. 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

36 2 Not applicable Not applicable P > 0.05

S. oregonensis

abundance No effect 6.4 μg/L Days 16 and

36 2 Not applicable Not applicable P > 0.05

Ceriodaphnia

sp. abundance No effect 6.4 μg/L Days 16 and

36 2 Not applicable Not applicable P > 0.05

D. pulex

abundance No effect 6.4 μg/L Days 16 and

36 2 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 abundan ce 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 an alyzed 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 pesticid es but does not report if the

concentration of ethanol in the at razine-only treatment was also 0.003%.

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the we ight of evidence regarding the potential for effects of atrazine on aquatic plant communities.

Limitations of Study: The results have good applicabil ity to mesocosms or natural aquatic systems because the experimental mesocosms were outdoors during testing and contained phytoplankton, pe riphyton, zooplankton, and two species of amphibians. Actual pesticide (atrazine) concentrations were not monitored throughout the experiment, although an initial measured value was reporte d. Control tanks were not analyzed for atrazine and the other nine pest icides 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, EPA169 3.42. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. Niedzw iecki, 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. Shephe rd, P.H. Crowley, T.M. McCarthy, J.H. Niedzwiecki, T. Sager, A. Sih, and B.D. Pa lmer. 2003. Lethal and Sublethal Effects of Atrazine, Carbaryl, Endosulfan, and Oc tylphenol 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 base d 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 th e presence and absence of food (carbaryl, endosulfan, and octylphenol were also examined). No effect on embryo or larval survival, hatching, or growth or developm ent (i.e., mass, SVL, and limb deformities) rates were observed at any of the test c oncentrations. Systema tically tapping tanks caused a greater activity in larvae exposed to 400 ppb atra zine, suggesting underlying nervous system malfunction according to the st udy author. Hunger stimulated a decrease in refuge use and an increase in activity (based on regres sion; no significant difference detected at any level); howeve r this response was least prono unced in the larvae exposed to atrazine at 400 ppb, possibly because th ese 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 presen ce 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 spri ng-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 grow th at highest test concentration.

Rationale for Use: Information on the t oxicity of atrazine to streamside salamanders Limitations of the Study: Solvent control contained bot h DMSO and acetone, whereas the atrazine treatment groups contained DMSO only. DMSO is not an acceptable solvent

because it accelerates the movement of a chem ical 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 a ppeared 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 collect ed so prior exposure history is unknown and the authors state that it is likely that they we re 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 temperatur e) and the loading was greater than the recommended 1 tadpole (larvae) per liter solutio n. Growth parameters were measured at Day 50 and analyzed. The stage of the organism s at Day 50 were not reported but if there was variability in the stages, it could potenti ally 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 Programs171 3.43. Rohr, J.R. and B.D. Palmer. 2005

Chemical N ame: 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 late r 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 terrari a 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 gr oups of three into petri dishes at a room temperature of 26°C (relevant mean summer soil temperature a ccording 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 obs erved water-conserving behaviors (moving and pressed against dish and number of or ganisms 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. Th ere was no difference be tween the earlier trial

and the later one for behavior or mass loss a nd 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).172173 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 so lvent. Organisms field collected and therefore prior exposure unknown. Study auth ors have previously reported that organisms likely exposed due to agricultu ral 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 wa s 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. Detailed174 descriptions on how the water conserving be haviors 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 respons ive 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 gr oups overlay it to the extent it is masked.

Reviewer: Amy Blankinship, Chemist, ERB3, Environmental Fate and Effects Division, Office of Pesticide Programs175 3.44. Rohr, J.R., T. Sager, T.M. Sest erhenn, and B.D. Palmer. 2006

Chemical N ame: Atrazine (Streamside Salamander St udy; 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. Hea lth 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 duri ng exposure was ameliorated by densitydependent survival after exposure, but complete density-dependent-mediated compensation was precluded by significant “carry over effects” of atrazine. Salamanders exposed to 4, 40 and 400 ppb of atrazine had si gnificantly lower survival than did control animals 14 months (421 days) postexposure. The study author atte mpts to predict population-level effects as the sum of exposure effects, carryover effect s (ie., effects of a pollutant may persist or app ear 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, Niedzwieck i 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 c ontained approx 6 cm of hom ogeneously 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 amphi bians. 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 em bryo, suggesting a fungal pathogen, which may

have caused decreased survival and increased time to hatching; effects on larval survival were different for different y ears (in 2002, there was no significa nt effect of atrazine on larval survival, whereas in 2003, larvae exposed to 400 ppb had significantly lower survival than control larvae). Metamorphi c parameters for 2003 included outliers and were not included in the analys es. Duration of study not spec ified (~117 days). DMSO is not an acceptable solvent becau se it accelerates the movement of a chemical across cell membranes. In addition, no negative contro l (water only w/out DMSO) was included.

Field collected organisms, therefore prior expo sure is unknown; however, in another study by Rohr et al ., 2003, at reported same collection s ite, they state that exposure from atrazine was likely given agricultura l 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, ERB3177 3.45. Rohr, J.R., A.A. Elskus, B.S. Shepherd, P.H. Crowley, T.M. McCarthy, J.H. Niedzw iecki, 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. Shephe rd, 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 Hydr operiod. 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 th e streamside salamander ( Ambystoma barbouri ) from the embryo stage through metamorphosis. In gene ral, food and atrazine levels did not interact statistically. Expos ure to 400 ppb atrazine decrea sed embryo survival to Day 16 and increased time to hatching. However, mo st 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 limitatio n 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 eff ects 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 reduc ing exposure, but their smaller size at metamorph could result in lower terrest rial survival, lower reproduction and compromised immune function. Drying condi tions accelerated meta morphosis 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 eith er factor alone. Food limitation, drying conditions, and 400 ppb of atrazine reduced size at metamorphosis without affecting body condition (relationshi p between mass and length), even though178 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 re lated to activity and feeding. Larson study showed that atrazine at 250 ppb altered thyr oxine 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 o C, 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 w ith 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 condi tions; 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 pr esence 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 c ontrol 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 th e movement of a chemical across cell membranes. In addition, no negative contro l (water only w/out DMSO) was included.

Organisms were field collect ed, therefore prior exposure history is unknown. The study authors state that they do not have data on atrazine concentrat ions at collection site they

state that an atrazine concen tration of 33.9 ppb was detected in KY stream similar in size179 to stream where organisms were collected Additionally, these or ganisms 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 ag ricultural practices surrounding the collection

site.

Reviewer: Anita Pease, ERB3; Amy Blankinship, Chemist, ERB3, Environmental Fate

and Effects Division180 3.46. Rohr, J. R. and P. W. Crumrine. 2005

Open Liter ature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Rohr, J. R. and P. W. Crumrine. 2005. Effect s of an herbicide and an insecticide on pond community structure and proce sses. 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 rega rding effects of atr azine 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 herbic ide atrazine and the insecticide endosulfan (tested independently) on constructed out door freshwater pond communities when the presence of common community members (w ood 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 we re hatched and reared in outdoor wading pools (containing aged tap water and covered with 60% shade cloth). They were fed

rabbit chow ad libitum. Clutch es were thoroughly mixed prio r 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 us e in the experiment. Late -instar dragonfly larvae ( Anax
junius ) were collected in early March 2003 from various ponds at the University of181 Kentucky’s Ecological Research Facility. Prior to the expe riment, the dragonflies were held individually in the laborator y and fed aquatic invertebrates and R. sylvatica tadpoles.

The study authors employed a 3 × 2 × 2 × 2 comp letely randomized f actorial 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 treatm ents (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 replicat ed four times in four spatial blocks (96 experimental units in total). No water (neg ative) control was incl uded. 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 th e 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, transparen t plastic cup (0.296 L capacity) secured to

one end of the tub with monofila ment line and five microscope slides attached to the side

of the tub with silicone. The tubs were loca ted on gravel at the Un iversity 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 surroundi ng 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, an d insect larvae/eggs. The tubs were covered with 60% shade cloth to prevent pred ation and allowed to mature for two weeks in order to establish a periphyton community pr ior to addition of the pesticide, tadpoles, snails, and dragonflies.

On April 14, 2003, the three “pesticide” treat ments (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 atrazi ne 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 st ock 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 simu late 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 concentrati on was not monitored during the experiment. However, the study authors estimated that th e nominal concentration was higher than the actual concentration due to heavy rainfa ll that occurred during the experiment.182 Tadpole, snail, and dragonfly masses were obtai ned 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 coul d 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 c ups 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 o ff a microscope slide. Chlorophyll a was measured using American Public Health Asso ciation 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 week ly 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 proporti on of tadpoles avoiding the c up (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 re moved only during the last week of the experiment. The experiment was ended on May 12 th for blocks 1 and 2 and on May 13 th
for blocks 3 and 4. At experiment termin ation, 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% solu tion of benzocaine and preserved in 10%

neutral buffered formalin for later determina tion of Gosner developmental stage. Water clarity was quantified by the study authors us ing a scale of 1 to 5. Chironomid larvae (dipterans) of the genus Polypedilum (periphytivores and detr itivores) and cladoceran zooplankton of the genus Daphnia (primarily Daphnia pulex ; phytoplanktivores) were

the most common invertebrates in the meso cosms. Their abundance was quantified on a scale of 1 to 5. The study authors proposed that their introducti on 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 st atistical analysis); th ese observations were

rare.

All statistical analysis was conducted using Statistica 5.5a, using the mesocosm as the experimental unit. Proportion data were a ngularly transformed. Covariates and block effects were included when significant. Three separate multivariate analyses of variance (MANOVA) were conducted to analyze ta dpole behavior (% hiding, % observed avoiding cup, % observed moving), tadpole gr owth and development (mass/tadpole and Gosner stage), and snail growth and repr oduction (mass per snail and egg masses per snail). Univariate analyses of variance (ANOVA) were subsequently performed on those183 parameters. DO and pH were regressed ve rsus tadpole growth and development (mass per tadpole and Gosner stage) and snail growth and reprodu ction (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 dr agonfly survival. Repeated-measures ANOVA

was used to evaluate the effect of pesticide treatm ents on chlorophyll a in the first and second half of the experiment. Treatment ef fects on the number of snails that died and the number of tadpoles that reached meta morphosis were eval uated using Poisson regression (PR; with a log link). Water clarity and the abundance of Polypedilum and Daphnia were analyzed using logistic regressi on for an ordinal multinomial distribution (OMR; with a logit link). Th e presence of other insects a nd crustaceans and dragonfly survival were evaluated using logistic regr ession for a binomial dist ribution (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 regres sion (PR, OMR, BR), the study authors performed Bonferroni-adjusted linear cont rasts 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/m 2 , mean ± SE) relative to the solvent control (6.575 ± 0.411 mg/m 2 , mean ± SE, Tukey’s hsd, P < 0.001).

Effects on Water Clarity

According to the study authors, water clarit y was greater in mesocosms with atrazine (OMR, P = 0.045), although it is unclear whet her the statistical comparison was made

relative to the solvent control or endosulfan treated mesocosms.

Effects on Insects and Crustaceans

Relative to mesocosms containing solvent cont rol, 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 ‘othe r’ 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 la rvae were alive (atrazine, 17.50 ± 2.43 days; endosulfan, 21.56 ± 1.96 days; solvent 22.13 ± 1.72 days; ANOVA, F 2,45 = 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 (a trazine, endosulfan, so lvent 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 sign ificant differences at P < 0.05 among treatments according to Tukey’s honestly si gnificant difference multiple comparison tests. Figure 2 from Rohr and Crumrine, 2005.185 Effects on Snails

Snail final mass was reduced by atrazine rela tive 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, endo sulfan, solvent control) and dragonfly treatments (presence and absence) on snail mass and (B) effect s of pesticide and snail treatments (presence and absence) on tadpole mass. Valu es represent means and SE of eight mesocosms, and probability186 values are for the interaction be tween displayed treatments wh en controlling for significant predictors [see Tables 1 and 3 of Rohr and Crumri ne (2005) for predictors]. Figure 4 from Rohr and Crumrine, 2005.

Figure 3. Effects of pesticide treatments (atrazine, endosulfan, solvent contro l) on (A) the number of egg masses deposited per surviving snail and (B) tadpole Gosner developmen t stage. Bars represent means (± SE) of 16 mesocosms. Different lowercase letters above bars reflect significant differences

at P < 0.05 among treatmen ts 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]. Figu re 6 from Rohr and Crumrine, 2005.

Effects on Wood Frogs

The study authors determined that the majo r 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 behavi or 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 contro l 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 co ntrol on tadpole final mass (Tukey’s hsd, P > 0.05, Figure 2 ).

Figure 4. Effects of pesticide trea tments (atrazine, endosulfan, solv ent control) on (A) the percentage of tadpoles hiding (measured as the percentage of total individuals that were not observed) and (B) the percentage of individual s 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 differen ce multiple comparison tests when188 controlling for significant predicto rs [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 obse rved 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/m 2 );

mean solvent, (6.575 mg/m 2 ).

Tukey’s hsd,

P < 0.001

Polypedilum

abundance

score ~24%

decrease 50 μg/L (maximum) 1 Unknown 2 Could not be

determined. Could not

be

determined. Figure 1

OMR, P =

0.004 Daphnia

abundance No effect

observed 50 μg/L (maximum) 1 Unknown 2 Could not be

determined. Could not

be Figure 1189 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 Unknown 2 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 > 0.05191 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 we re 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 associ ated 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 pr esented by the study authors were not specific to atrazine treatment effects. Results of MANCOVA, ANCOVA, ANOVA, a nd Poisson regression did not distinguish the individual effects of atrazine. In gene ral, these analyses examined the effects of “pesticide” treatm ent (atrazine, endosulfan, or so lvent 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 re sults, that is, a pairwi se comparison between

atrazine treated and solven t control mesocosms. This study review focuses on and presents results specific to atrazine treat ment effects, unless otherwise noted (i.e., analyses and results that include endosulfan treatments are generall y 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 inte grated into the atrazine treatment effects presented in this study.

The study authors stated that di rect effects of atrazine ( i.e., reduction of periphyton chlorophyll a ) resulted in indirect eff ects 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 applicabil ity to mesocosms or natural aquatic systems since the experimental meso cosms contained not only periphytic algae, but also several species from higher leve ls of the food web, however, there were limitations. Actual pesticide (atrazine) con centrations were not monitored throughout the experiment. Although a nominal dose was re ported, exposure was complicated because a second dose of 25 μg atrazine/L was made tw o weeks after the ini tial application of 25 μg/L and the study authors re ported heavy rainfall (timi ng not reported) during the experiment which would have diluted the pesticide in the mesocosms. Nominal

concentrations were verified in the stock solution and atrazine s hould have been stable over a period of two weeks; therefore, th e 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; e ffects of acetone would have been underestimated if a second applicati on 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 acclim ation period for the snails, tadpoles, and dragonflies. In addition, it is unclear whet her 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, EPA193 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.

Piw oni, 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 rega rding effects of atr azine 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 num ber of trematode infections in the declining northern leopard frog Rana pipiens by studying the eff ects of atrazine on R. palustris and R. pipiens in prepared community-l evel mesocosms and by conduc ting 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 experiment194 The experiment was conducted at Pennsylva nia State University using a randomized block design. The Pennsylvania State Univer sity 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 (atrazi ne = 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, pe riphyton samplers, and inoculations of zooplankton, periphyton, and phytoplankton from four ponds (location of ponds not reported). Just before dosing, each test tank wa s 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 declini ng northern leopard frog R. pipiens . All snails which were used in the experiment were screened twice for pa tent (shedding) trematode infections before being placed in the tanks. Tanks were exposed to atrazine via a si ngle dose of technical grade atrazine (from ChemServ ice; % 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 usi ng the U.S. EPA’s GENEEC (version 2) software. One hour after dosing, water samp les were taken and shipped on ice for analysis of actual applied atra zine (mean 117 μg/L). In or der to simulate exposure to trematodes, each tank also rece ived a caged planorbid snail shedding cercariae from the family Plagiorchidae (the timing of this a ddition 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 numbe r of tanks per control) . The solvent used

and its final concentrat ion were not reported.

At the end of the experiment, the study au thors took water sample s for phytoplankton analyses, scraped periphyton fr om the periphyton samplers, and scored water clarity on a scale of 1 to 5. Additionally, the study aut hors 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 sp ecified). Five preserved R. clamitans and five R. palustris tadpoles from each tank were selected ra ndom ly and melanomacrophage and eosinophils cells were counted per field of view from th eir sectioned and staine d 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 fo r statistical analysis.

The effect of atrazine on chlorophyll a concentration in phytoplankton and periphyton and on percent survival (after angular tran sformation) was analyzed using the general195 linear model. Log eosinophils and log melanomacrophages per field of view and trematode loads were analyzed using the ge neral linear model cont rolling 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 demonstrat ed 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 th e generalized linear model with an ordinal multinomial error distribution and a logit link. The physid snai l 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 probabi lity value was less than 0.25. To determine if eosinophils and melanomacrophages were sign ificant predictors of plagiorchid loads, the study authors conducted a regression analys is 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 auth ors did not specify the statistic s 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 tr ematodes) at the beginning of the experiment and began shedding plagiorchid cercariae durin g 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 th e Broadleaf Forest Ecoregi on in Minnesota, USA, were surveyed regarding more than 240 plausibl e predictors of the abundance of larval trematodes in northern leopard frogs ( R. pipiens ) living in those wetlands. During 1999, the study authors characterized biological dive rsity (three visits: March-April, May-June and, July-August) and obtained water, se diment, and amphibian tissue samples to quantify various analytes (two visits: April- May and June-July). Analyte quantification was conducted using standard US EPA protocol s. During each visi t 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 sampli ng 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. La ndscape structure was summarized at 1-km and 10-km extents using Fragstats. Frogs were inspected for parasites (buccal and abdominal cavities, visceral organs, ski n, and eyes). After necropsy, a dissecting microscope was used to identify encysted metacercariae. Frog livers (frogs in Gosner stage 46) were examined for melanomacropha ge 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 (R 2 ) of each predictor in the ab sence of covariates; 2) used the general linear model to generate the 20 be st subset models for mean larval trematode

loads in R. pipiens (maximum of 3 predictors, ranked by R 2 ); 3) used a structural equation model, followed by path analysis to evaluate the amount of support for the specific proposed casual mechanisms underlyi ng the significant positive relationship

between atrazine concentrati on and larval trematode abundanc e; and 4) assessed whether atrazine was correlated with any other vari ables that might explain the results more concisely. Atrazine and desethylatrazine (one of the transf ormation products of atrazine) were measured separately; however, they were combined together as “atrazine” for the analysis since their combination was the be st predictor of total larval trematode

abundance in R. pipiens

.

Results:

Mesocosm Experiment

In the mesocosm experiment, atrazine si gnificantly reduced phytoplankton chlorophyll a concentrations (mean ± s.e.m.; atrazine treatment, 2.52 ± 0.45; water and solvent controls, 7.14 ± 1.67; ge neral 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 alg ae, however, only data on water clarity are reported ( Table 1 ). The concentration of periphyton chlorophyll a was significantly

elevated in the atrazine-treated mesoco sms (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 ta nks 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 atr azine-treated tanks (47.5% survival in the atrazine treatment group versus 70% su rvival 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 Atrazine 1 Control 2 Test statistic P 3

Chlorophyll a in phytoplankton (μg/L) 2.52 ± 0.45 7.14 ± 1.67 4.88 4 0.031 5

Chlorophyll a in periphyton (mg/m 2 ) 1.04 ± 0.18 0.70 ± 0.08 7.17 4 0.032

Water clarity (scale 1-5) 4.75 ± 0.25 3.88 ± 0.23 5.25 6 0.011 5

Snail hatchings (per m 2 ) 468.09 ± 205.85 110.64 ± 29.31 20.19 7 <0.001

Snail egg masses (per m 2 ) 30.45 ± 3.03 12.37 ± 4.34 4.98 7 0.026

Melanomacrophages per field

of view for R. palustris (log) 0.16 ± 0.06 0.36 ± 0.06 4.50 4 0.031 5

Eosinophils per field of view

for R. palustris (log) 0.03 ± 0.14 0.21 ± 0.06 1.88 4 0.102 5,8

Survival of R. palustris (%) 47.50 ± 7.50 70.00 ± 3.90 9.77 4 0.017

Melanomacrophages per field

of view for R. clamitans (log) 0.25 ± 0.13 0.38 ± 0.09 0.67 4 0.217 5,9

Eosinophils per field of view

for R. clamitans (log) 0.11 ± 0.10 0.39 ± 0.07 4.75 4 0.029 5

Survival of R. clamitans (%) 76.25 ± 3.15 76.25 ± 2.80 <0.001 4 0.971 10

No. of plagiorchid trematode cysts in R. clamitans 20.61 ± 4.52 7.51 ± 3.30 4.90 4 0.031 5,11

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 χ 2 statistic from analyses using the generalized linear model with an ordinal multinomial error distribution and logit link.

7 χ 2 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 ( F 1,7 = 5.45, P = 0.026) and mean log eosinophil counts in tadpoles ( F 1,7 = 3.58, P = 0.050).

Wetland Survey198 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 tota l larval trematode abundance in R. pipiens out of more than 240 measured plausible predictors of amphibian trematode infe ctions. Atrazine alone ( R 2 = 0.46, F 1,15 = 12.77, P = 0.003, see Figure 1 ) and desethylatrazine alone ( R 2 = 0.46, F 1,15 = 12.93, P = 0.003) were significant predictors of increased trematode infections in R. pipiens . The sum of atrazine and desethylatrazi ne accounted for more variation than any other single predictor (51%, M onte Carlo randomization test, P = 0.001; see Figure 2a ). Atrazine combined with desethylatrazine was a significant negative predictor of melanomacrophages aggregate scores ( R 2 = 0.17, F 1,16 = 3.17, P = 0.047; See Figure 3 ). Melanomacrophage scores were from 0 (not pr esent) to 5 (> 90% of liver filled with melanomacrophage aggregates).

Figure 1. The relationship between larval trema tode 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 fo r the relationship between atrazine and larval trematodes in amphibians. a, The relationship between detectable co ncentrations 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 stan dardized coefficients, respectivel y, 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 residual s from the relationship among me an melanomacrophage aggregate

scores and the richness and abunda nce of nematodes and adult tremat odes 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 tr ematode loads in amphibians must depend on the abundance of definitive hosts, such as am phibian-eating birds (a source of trematode eggs in wetlands). This dependence was s upported by the significant interaction between

atrazine and the number of suitable host habi tat patches (forest, wetlands and open water) around each wetland (Monte Carlo random ization 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. Howe ver, in the supplemen tal information (found

online at http://www.nature.com/nature/jour nal/v455/n7217/extref/n ature07281-s1.pdf ), some results were presented for atrazine a nd desethylatrazine indi vidually. In addition, supplementary details about the models and resu lts 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 prim ary driver of larval trematode infections for a declining201 amphibian species at concentrations commonly occurring in freshwater ecosystems (they also suggested that phosphate played a comp lementary role). The study authors proposed that atrazine increased exposure and suscepti bility to larval trem atodes by increasing the

amounts of snail intermediate hosts (via increasing the peri phyton) and by suppressing amphibian immune responses. The scheme of the proposed path of atrazine to increased

larval trematode infections and mortal ity in amphibians is illustrated in Figure 4

.202 Figure 4. A simple scheme which summarizes the ma in 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 = 0.031 2

Chlorophyll a in periphyton 49% higher 102 μg/L Four weeks, study

termination. Could not be

determined. Could not be

determined. P = 0.032 2

Snail hatchings 323% higher 102 μg/L Four weeks, study

termination. Could not be

determined. Could not be

determined. P < 0.001 2

Snail egg

masses 146% higher 102 μg/L Four weeks, study

termination. Could not be

determined. Could not be

determined. P = 0.026 2

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 = 0.031 2

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 = 0.102 2

Survival of R. palustris 32% lower 102 μg/L Four weeks, study

termination. Could not be

determined. Could not be

determined. P = 0.017 2

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 = 0.217 2

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 = 0.029 2

Survival of R.

clamitans No effect 102 μg/L Four weeks, study

termination. Could not be

determined. Could not be

determined. P = 0.971 2

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 = 0.031 2

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 prov ided regarding the water used for the mesocosm experiment (t he pond characteristics and locations of the four ponds were not specified; freshwater sour ce 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 pest icides) prior to the e xperiment, given that no previous contaminant history or the loca tion of the collection sites was provided.

There was a three week acclimation pe riod for zooplankton, phytoplankton, and periphyton; however, all other taxa were added to the tanks just before dosing with

atrazine.

Atrazine concentrations were not monito red 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 pond s or the control mesocosms.

It was not reported if the alga l communities (i.e. chlorophyll a concentrations) were similar among the mesocosms prior to dosing.

The generalized linear model was used to te st for significant differences between the

atrazine treatment and the cont rols. The study authors did no t explain why this analysis was chosen instead of a simpler statistic al method for comparing two treatments.

The study authors suggested that the potentially important re lationship between atrazine (pollution) and amphibian parasi tism would not have been det ected in the standard tests

conducted in the United States and Europe for chemical regi stration since the tests are typically conducted on solitary species (isola ted 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, wate r bug, and dragonfly species); there

were a number of limitations associated with the study. The study au thors 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 uncle ar when the analysis occurred. The source ponds and the control mesocosms were not an alyzed 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 th e 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, EPA206 3.48. Rohr, J. R., A. Swan, T.R. Raffel, and P.J. Hudson. 2009.

Open Liter ature 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-disr uption, and the ecology of fear. Ocecologia . 159:

447-454. (ECOTOX Ref. No. 11715)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 perc eption (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 co ntrol (0.0002%) (n=6 cups) for 4 days under static conditions. Tadpoles held at 20°C and fe d fish flakes and frozen spinach for first 3 days and no food on 4 th and after 4 days survival was re ported at nearly 100%. Tadpoles were collected shortly after hatching fr om 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 res ource); or two macerat ed conspecifics (predation cue). All 12 treatments were te sted each day; ten experimental blocks; no tadpole was tested more than once. Behavior al 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 ta dpoles. Three tadpoles removed from the cup (placed into a nitex cage) for each gutter/tr ial and tadpoles allowed to swim freely in gutter; authors state allowed for 10 replicat es/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 pl aced into one end of a gutter with an empty cage at the207 other end of the gutter. Black window screens placed in front of cages to prevent tadpoles from seeing cues orhiding 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 ex posed to cues for one hour and from the video, the location of each ta dpole was recorded every 2 minut es which the author stated was to evaluate whether tadpole was attracte d to, or avoided the cues. The number of lines the tadpole crossed was also measured; an average distance a nd number of lines were used in the statistical analysis.

Results

Measured concentration of at razine stock was 196 μg/L. The study authors state that tadpoles were significantly cl oser to food cages (p=0.026) and significantly avoided [by distance from cage/cue] macerated conspeci fics (p=0.004), a snail shedding cercariae (p=0.022) and cercariae alone (p=0.004) but did not avoid [b ased on distance] uninfected snail. There was no significant difference be tween responses for inf ected snail, cercariae alone, or macerated conspecifics. In additio n, previous exposure to atrazine did not significantly affect responses for any of the cues. Cercariae alone and infected snail treatments significantly increas ed tadpole activity (by numb er of segments crossed) (p<0.044); but other treatments not significantly different including pr evious 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 provide s 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 stud ies 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 ot her chemicals present. Analysis of food source for potential contaminants not reporte d. Other than temperature, water quality parameters were not reported; test vessel ma terial not reported. A lthough the study author states that previous work showed no effect fr om solvent, there is so me 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 se ven snails were used in the expe riment. Also the mean number

of cercariae released was measured for tw o 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 review er 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 uncertain ty. However, it is possible th at the tadpoles were exposed

to different numbers of cercari ae [or amount of chemical cue] each day and it is uncertain how that may have impacted behavior.

The authors acknowledge the difficulty in st andardizing 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 applicable209 3.49. Rohr, J. R., T. M. Sesterhenn and C. Stieha. 2011.

Open Liter ature 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 susceptibil ity (adverse effects from) to atrazine.

Methods

Wild collected salamander eggs (Harrison st age 18-28) were separated and then 12 eggs (embryos) each placed into 64 glass bowls contai ning 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) co ntrol 165 mL/2 L (8.25%) was used; however, no negative control since [accord ing to the authors] similar solvent concentrations were shown to have no significant effect. Study was conducted using st atic renewal with complete water changes once per week.

EMBRYO STUDY

For the embryo studies, two environmental chambers were set at 13 o C and two were set

at 19 o C; 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. Bo wls were checked daily for hatching and mortality; larval mass was determined from formalin-preserved specimens

L ARVAL S TUDIES

Larval studies were conducted using 4 repli cates for each atrazine concentration within

each of the 4 environmental chambers set at different temperatures: 16, 19, 22 and 25 o C.210 Two of the replicates of each atrazine concentration in each chamber were transferred to water containing acetone alon e once the first larva metamo rphosed, 19 days into the experiment (from the 25 o C chamber); therefore, half of th e 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 so lvent 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 di d 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 si gnificantly higher at 19

than at 13 o C, the authors state that salamanders hatched sooner ( Figure 1c ) at 19 o C.211 L ARVAL S TUDIES

According to the study aut hors, atrazine significan tly 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 nega tive predictor of larval survival" and they site their previous studies showing that atra zine significantly reduced survival of larval A.

barbouri and had one-tailed statis tical 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 sma ll in comparison to the positive effects of
increased temperature by accelerating the sa lamanders' development thereby reducing
exposure to atrazine (larval study).212 The authors conclude that increasing atrazi ne concentrations reduced growth, delyaed
hatching and metamorphosis, and decreased embr yonic 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 e ffects 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 gene ral that there was much higher mortality of embryos and post-hatch larvae at

19 o C. Embryos at 19 o C took less time to hatch (13 - 14 days) and those reared at 13 o C (27 - 31 days); in spite of the increased time to hatch, embryos had a lower mass at hatch compared to embryos reared at 19 o C.

The larval study demonstrat es that increasing water temperature from 16 to 25 o C decreased the length of time to metamorphosis; although the increase in water temperature from 22 to 25 o 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 o C appears to have been associ ated with reduced mass of the metamorphs.

Limitations of Study:213 Wild-caught animals; therefore, prior exposur e history is unknown. E ggs were separated; however, it is uncertain whether this invo lved de-jellying the eggs and how this separation effort may have influenced exposur e. No negative control; authors assumed

that the co-solvent would not affect study (based on histor ical data). Developmental stage of the test animals is not well characteri zed 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 figu res. Poor larval survival across acetone

control and atrazine treatme nts (roughly 60%; depicted in Figure 1b ) suggests that survival was poor across all of the animals in the 19 o C treatment and as such, it may be

difficult to make inferences about ot her treatment effects in this group.

Primary Reviewer:

Thomas Steeger, Ph.D., Senior Science A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable214 3.50. Spolyarich, N., R. Hyne, S. Wilson , C. Palmer and M. Byrne. 2010.

Open Liter ature 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, devel opment 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 2 nd 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 (Gosne r 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 a nd was on a 12:12 light:dark cycle. Exposure commenced just prior to gonadal differen tiation (Gosner stage 30). Animals were removed at Gosner stage 44 or 45. Spiki ng took place after every water renewal after every 3 rd or 4 th day of exposure. DO, temperatur e and pH were measured daily. Table 1 (reproduced from report) summarizes the variou s treatments. Sex ratios initially based on gross gonadal morphology. Male gonads from Tests 1, 6, 7 (atrazin e 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 thi obencarb were detected in cont rols. 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 showi ng that they did not vary from controls by more than one or two stages at any time pe riod. No significant differences were found between the treatments and controls for any test.

Atrazine, metolochlor and thiobencarb did no t 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 lengt hs of larvae exposed to atrazine of 0.1, 3 and 30 μg/L over 4 weeks compared to c ontrols (Test 7). This authors noted that this particular test produced the largest although not significant differe nce 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 exposur e; the authors attributed this to handling

stress. There was no evidence of a concentra tion-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 obser ved in 1.9% of the animals and was again randomly distributed among treatments and controls.

Combining controls across tests, the sex ra tio was 50.3:49.7 male:femal e. According to the authors, none of the sex ra tios 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 Te st 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

testes217 The authors conclude the exposure of spotte d march frog lavae to atrazine (0.1, 1, 3 and 30 μg/L, metolachlor (0.1, 1 and 10 μg/L) and thiobenc arb (90, 180, and 360 μg/L) from Gosner stage 28 to 42 under controlled lab c onditions had no signif icant effect on tadpole growth, development and sex ratio. Binary mixt ures of atraine and thiobencarb as well as ternary mixtures of all three also had not significant eff ect on the devel oping 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 at razine, metolachlor a nd thiobencarb both singularly and in combination on the spotte d marsh frog. Studies of atrazine were reproduced three times with relatively consistent results for sex ratios.

Limitations of Study:

Wild-caught animals; therefore, prior e xposure history is unknown. Histology was only conducted on male gonads. Sex was appa rently based on gross gonadal morphology

alone and was not verified across all of the te st animals with histol ogy. Given the n value (3), it is presumed that the gra phs for mean rate of development ( Figure 1 ) and mean length ( Figure 2 ) represent pooled data from the th ree 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 he rbicide mixtures. In addition, based on the figures, it appears that psueor eplication occurred as the sample size for each treatment is 30 (total individuals exposed per treatment) as oppose to ‘n’ of 3 (t hree replicates/trials218 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 th e ‘n’ to 3 is expected to give the same result.

Primary Reviewer:

Thomas Steeger, Ph.D., Senior Science A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable219 3.51. Storrs, S.I. and J.M. Kiesecker. 2004

OPEN LITERATURE REVIEW

Chemical N ame: 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. Su rvivorship 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 surv ivorship; survival was significantly lower for all animals exposed to 3 ppb compared w ith either 30 or 100 ppb, except the late stages of the toad and wood frog. There we re 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 signi ficant 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 c oncentrations of atrazi ne died sooner that animals exposed to medium or high concentr ations, 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 pol ypropylene 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. P hotoperiod = 12 h light/dark; temp = 22 o C. Animals fed crushed alfalfa ever y 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 co nsistent response.

Limitations of the Study: No raw data provided. Time to significant mortality (relative to the control) was not discussed. Animal s 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 othe r 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 Programs221 3.52. Storrs Méndez, S.I., D.E. Tillitt, T. A.G Ritten house, 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 Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 Ameri can 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, B oone County, MO) were placed into test

chambers that contained control soil as we ll 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-s outh 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. Bo th nights included all treatments, each toad used only once, and after toads were weighe d they were acclimated to chambers and toads were observed with a red-light headlam p. 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 fi rst hour and then every 12 hours for 2.5 days. For the uptake and elimination test, 17 toad s 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 atrazi ne). Food was withheld for 72 hours prior to initiation and toads were placed in empty test chamber ove rnight to allow dehydration. An attempt to empty bladders prior to test to obtain st andard mass was made but 10 out of 17 animals did not empty bladder. Location of the pelv ic 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 a nd replaced with clean water to examine222 elimination. Organs were removed and measur ed 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 toad s 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 radioactiv ity in water at between hour 4 and 8 and decrease after hour 8 water change. The greatest radioactivity pe r 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 intes tines at hour 8 [Figure 4 indicates the peak was at hour 12 for both tissues]. Peak concentr ations 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 quantita tively linked to the endpoints of survival, growth and reproduction used in a risk assessment. While the study evaluated movement from atrazine treate d water, statements about potential uptake while traversing atrazine contaminated soil can not 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 th e 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 degr adation during storage or not.

According to the study, the atrazine stocks fo r 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 exte nt the solvent influenced the uptake of the

atrazine is not known.

As 10 out of 17 animals did not relieve thei r bladders, the goal of trying to obtain a standard mass was not achieved for every anim al and there is some uncertainty in how this affected subsequent mass analysis.223 According to the authors, one gall bladde r burst during dissecti on 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 sele cted toads from the so il 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 experime nt may have affected the uptake study.

Total radioactivity was measured in the uptak e study with no further analysis to measure metabolites. Therefore, comparisons among or gans and timepoints within an organ are based on the assumption that the radioactivity m easured is parent atrazine when in fact it may be a combination of differe nt 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 applicable224 3.53. Storrs-Méndez, S. I. and R. D. Semlitsch. 2009

Open Liter ature 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: Unde rstanding the Time Course of Natural Development and Role of Endocrine Disruptor s. Journal of Experimental Zoology (Mol Dev Evol) 312B: 1 -11. (ECOTOX Ref. No. 153841)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 suscep tible to endocrine disruption as larvae and metamorphs (juveniles) than species with sl ower rates of sexual differentiation. The goal of this study was to determine whether th is susceptibility has an effect on gonad morphology in terrestrial anurans no longer expo sed 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 exhibiti ng 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 ha ve an accelerated rate of ovary development.

Methods

Larval studies examined three atrazine concen trations (1, 3 and 30 μg/L), ethanol control

and a 17- β

estradiol (10 -7 M) control. The study was c onducted 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 whic h the treatment units were located in the laboratory. Each replicate cons isted 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 meta morphosis (complete tail resorption [Gosner stage 46]) were sacrificed for assessment of gon adal histology to asse ss the proportion of225 males, females, underdeveloped testes UT), testicular oocytes (TO; testes with 0 – 30% oocytes) and ovotestes (OVT; te stes 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 meta morphosis were record ed. For all of the test animals, the kidney-gonadal complex was pr epared for histological analysis. Studies

with B. americanus were conducted 1 year after the st udies with the other two species.

Juvenile studies examined two atrazine con centrations (1 and 10 μg/L), solvent (ethanol) control, and 17- β

estradiol control. A ccording 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-gona dal complex of the test animals were prepared for histological analysis.

Results

Treefrogs were raised for 124 ± 0.15 (mean± std err) days after larv al exposure started; leopard frogs were raised for 120 ± 0.05 days an d 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 leopa rd frogs were conducted in

Table 1. Summary of measured exposure co ncentrations 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 co ncentrations 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 atra zine 30 μg/L compared to controls and that226 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 ethano l control toad did not differ significantly from estradiol controls (Figure 1A); however, for treefr ogs, sex ratios in the ethanol control were signif icantly different (lower) than the estradiol control

Figure 2 (reproduced from the stud y) 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 et hanol control; at 9 wks there we re 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 (r oughly 27%) compared to estradiol-treated frogs with roughly 50% based on Figure 2C . For leopard frogs, th e sex ratio of ethanol controls was only significantly different than estradiol controls at 6 wks ( Figure 2B )227 Figure 3 (reproduced from the report) de picts 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 gonada l 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 am ong 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 underde veloped 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 concentratio ns 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 th eir prior exposure history is unknonw. No negative control was run. S ource of the dilution water not stated. Although not clearly articulated, the larval studies appear to ha ve been conducted in gl ass containers until larvae were close to metamorphosis when th e 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 ex perimental 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 th at 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 sect ion how juveniles were exposed to atrazine,

i.e ., whether exposure was just during their larv al develop period or whether they were somehow exposed during the juvenile period as well.

The authors do not report whether mort ality was an issue in the study.

The limit of detection (LOD) for atrazine an d estradiol are not re ported; the limit of quantification (LOQ) for atr azine is 0.05 μg/L. In the B. americanus study, atrazine was

detected in the estradiol cont rol at 0.05 ppb. Measured concen trations 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 concen trations 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 at razine was conducted throughout the larval study period, it is unclear when measur ed concentrations were recorded.

The authors report that kidney-gonadal complex was sectioned longitudinally into 7 μm sections; however, it is not stated wh ether 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 rela tively 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 r oughly 29% female by metamorphosis ( Figure 2C ). The changing sex ratios suggest that gonadal development was inconclusive prior to metamorphosis and does not serve as re liable /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 th e toad, the number of females was higher [as expected].

Figure 5D of the paper (not repro duced in this DER) reports results from 10 μg/L atrazine treatments form both larval and juve nile experiments; however, larvae were not exposed to 10 μg/L.

The authors note that the number of testicular oocytes were higher ne ar the proximal end

of the testes of toads where th e 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 gene rally not well characterized a nd it is unclear whether the Bidder tissue is actually infiltrating (bleeding in to) 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 underdevelope d 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 es tradiol-treated amphibians. Also, UT did not appear to be dose-responsive nor did it co nsistently appear in the estradiol-treated

animals.

Primary Reviewer:

Thomas Steeger, Ph.D., Senior Science A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable230 3.54. Wan M.T., C. Buday, G. Schroeder, J. Kuo, and J. Pasternak. 2006

Chemical N ame: Atrazine

CAS No: 1912-24-9

ECOTOX Reference Number and Citation: 89626

Wan M.T., C. Buday, G. Schroeder, J. K uo, and J. Pasternak. 2006. Toxicity to Daphnia magna, Hyalella azteca, Oncorhynchus kisu tch, 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 (A trazine 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 th e table below (reprint from the study).

The acute toxicity for technical and atrazine formulation ar e 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 effect s 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, ERB3232 3.55. Williams, B. K. and R. D. Semlitsch. 2010

Open Liter ature 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 Anur ans to Chronic, Low-Dose Exposures of

Four Herbicides. Arch. Environ. Contam. Toxicol. 58: 819 - 827 (ECOTOX Ref. No. 153825)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 expos e three common species of tadpoles to conservative levels of atrazine, S-metol achlor, 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 25 o C 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 c oncentrations 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 pe r treatment under stat ic 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 t ypically took an average of 3 days.

Results

Mean-measured concentrations ( over three time points in the st udy) 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 toad s and chorus frog surv ivors metamorphosed; whereas 26% of the surviving treefrogs ha d 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 expos ed to glyphosate at 572 μg a.e./L; larvae exposed to Roundup ® WeatherMax metamorphosed 14% late r (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 marg inally 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 meta morphosis. 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 wester n chorus frog larvae that was likely the result of a "unique surfact ant" 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, prio r exposure history is unknown. Source of the

dilution water not stated. Concentrations of glyphosate and S-me tolachlor were not

measured and are based on nominal concentr ation only. Control survival (roughly 80%) was relatively low for chorus frogs.235 Primary Reviewer:

Thomas Steeger, Ph.D., Senior Science A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable236 3.56. Zaya, R. M., Z. Amini, A. S. Whit aker, S. Koh ler 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 expos ure affects growth, body condition and liver

health in Xenopus laevis tadpoles. Aquatic Toxicology 1 04: 243 – 253. (ECOTOX Ref. No. 153847)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 metamor phic rate, and a reduction in body weight and length and fat body size) noted in a previ ous 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 a ssess the cellular mechanisms these phenotypic effects in exposed African clawed frog ( X. laevis ) larvae.

Methods

Six separate experiments were conducted: 4 st udies (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 st udy (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 50 o C for no more than 15 min). Five days post hatch (NF stage 47 larv ae separated into bow ls containing 1 L of spring water with and without at razine: (6 larvae in Studie s D and E), 8 (Studies A and C), 10 (Study F) and 22 (Study B); after 2 w eeks, bowls had a fina l 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 remai nder of the study. Bowls were stacked on laboratory shelves and relied on room temperature (19 – 23 o C) and ambient light. Exposure water was completely replaced th ree 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 stag ed. Appetite was monitored in three studies between 8 – 8:30 am 3 to 5 days per week by observing behavior patt ern. Depending on the experiment, larvae were sacr ificed 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. Fat237 bodies were photographed. Livers and fat bodi es were then prepared for histology. Differential staining for glycogen and caspase-3

Results

Measured concentrations of atrazine in th e he stock and 1 μg/L concentrations were within ±4% of nominal; however, neither of these represents actual treatment

concentrations. Accordi ng to the study authors, none of th e 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 exhi bited significantly (p<0.03) lo wer 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 signif icantly 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 between238 controls and atrazine-treated an imals 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 base d on gross gonadal morphology compared to controls. There were no intersex animals noted in any of the atrazine-exposed animals; howev er, histology was not conducted.

There were no statistically si gnificant 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) lo wer (15%) than controls. In terms of length, none of the larvae expos ed 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 rela tive 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-treat ed and control larvae; ther e was no difference in fat body lipid content between atr azine 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 a nd 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 include d reduced liver and fat body sizes and that the changes indicated that exposure to atra zine throughout larval development “compromised” the larvae and potentially decrease d their ability to surv ive the stresses of metamorphosis or reduce reproductive fitness si nce frogs rely on stor ed lipids for these

purposes.

Description of Use in Document (QUAL, QUAN, INV):

Qualitative

Rationale for Use:

Although the concentrations test ed in this study are of uncer tain ecological relevance and although the studies were sepa rate, the studies provide info rmation that mortality and gonadal development (based on gross morphol ogy) were not affected at atrazine concentrations up to 400 μg/L. The statistically signi ficant effects (reductions) on body weight and on fat body and liver weights expressed as percent body weight did not appear to be dose-resp onsive and did not appear to resu lt in any abnormal histological effects on the organs. The activation of caspa se-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 dens ity 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 conditi ons. Also, reported environmental conditions suggest that water temperatures fluctuated broadly (19 – 23 o C) and that lighting was not uniformly distribut ed across the treatment units. Removing all of the larvae from each exposur e to record collective weights is likely a major source of

stress for the test animals and it is uncer tain 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, th e results section sugge sts that several of the studies measured individual weights of the test animals; collective weight240 measurements would be stressful, but indivi dual weight measurements would likely have increased the amount of handling and conseque ntly stress. Stag ing 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 a ssessed. 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 re ported in surface water adjacent to field tr eated with atrazine.

It sounds as though NF Stage 47 was select ed as this would correspond to when Xenopus

laevis larvae would initiate f eeding. Exposure proceeded to NF Stage 62; this would correspond to metamorphosis when it is likel y that test animals w ould cease feeding and begin to resorb tissues to sustain the metamorp hosis 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 afte r acute exposure to 400 μg/L; however, since exposures were conducted for several weeks, th ey could not be considered “acute”. The authors report "near-significant" results; howev er, such observations have little utility as there is no way to determine short of additi onal 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 si gnificantly shorter. Although all of the animals exposed to 400 μg /L weighed signifi cantly 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 di d 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 m easurement endpoint; in one to the two studies conducted at 200 μg/L and half of the stud ies 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 significan t measurement endpoint from histology was that larvae exposed to 400 μg/L had liver sections with significantly grated number of activated241 caspase-3 hepatocyes. The fact that this fi nding is limited to only one of the four studies which examined this exposure concentration li mits its utility especially given that no

other abnormal effects were reported through histology.

The authors’ conclusions that exposure to atrazine throughout la rval development “compromised” the larvae and potentially decrease d their ability to surv ive the stresses of metamorphosis or reduce reproductive fitne ss is speculative and ca nnot be supported by

the data contained in their study. Although the re searchers 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 A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable242 3.57. Zaya, R. M., Z. Amini, A. S. Whitaker, and C. F. Ide. 2011

Open Liter ature 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 aff ects the expression of key genes in metabolic pathways integral to energy homeostasis in Xe nopus laevis tadpoles. Aquatic Toxicology 104: 254 – 262. (ECOTOX Ref. No. 153848)

Purpose of Review (Note: DP Barcod e required for Quantitative studies):

Determine the relevancy of the study for eval uating 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 th e hypothesis that atrazi ne exposure altered energy homeostatsis in Xenopus laevis tadpoles. The authors re lied on an reanalysis of Affymetrix microarray data generated in Langerveld et al ., 2009 and included quantitative real-time polymerase chain r eaction (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 . 2011 77 (ECOTOX Ref. No. 153847; DER co mpleted 11/19/11). Tadpoles

were obtained from in-house br eeding colony. Larvae (5-day post hatch; NF stage 47)

stocked at a rate of 8 larvae/L until two week s 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 – 23 o C with ambient light exposure; water was completely replaced 3 times weekly and animals were fed a suspen sion of powdered Purina rabbit chow (#5354)

ad libitum . Exposure to 0, 200 or 400 μg/L con tinued from Day 5 post-hatch (NF stage 47) for approximately 50 days until NF stage 62.

GeneSifter TM , an on-line gene analysis software tool was used to determine which metabolic processes were “over” or “under-re presented” in the overa ll gene expression observed through the Affymetric microarr ay analysis reported in Langerveld et al . 2009. Twelve chp files from control and 11 chp file s from the 400 μg/L exposed larvae were

77 Zaya, R. M., Z. Amini, A. S. Whitaker, S. Kohler an d 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 as says; 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 duplic ate was calculated and only those duplicates

with a standard deviation ≤ 0.25 were used for data anal ysis; gene expression was normalized to the internal yeast standard.

Larvae from Study F were collected at 24 (c ontrol=10; 200 μg/L=9; 400 μg/L=11), 48 (n=10 for all groups) and 72 hr (control=11; atrazine=10) of expos ure); additionally, one tadpole from each bowl was collected at NF stage 62 (control=12; atrazine=8) and then weighted, length measured and its liver ex cised for ATP and ADP analysis. A known amount of ATP was spiked into a sample ali quot to determine recovery; recovery of ATP

was >98% and it was assumed that there wa s 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 de termined); ATP alone was measured using

an ATP Determination Kit. The ratio of ADP:ATP could then be determined.

Results

Based on the Affymetric/GeneSifter anal ysis, up-regulated genes included those associated with the synthesis and degradation of ketone bodies, the urea cycle, regulation of autophagy, citric acid cycle, glutathi one metabolism and metabolism of a number of amino acids (methionine, alanine, aspartate, arginine, proline, glycine, serine, and threonine. Down-regulation included glycolys is/gluconeogenesis, fatty acid elongation in mitrochondria and ribosomal proteins.

The authors note (based on their previous public ation) 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 co llected at 24, 48, 72 hrs a nd 14 days after initiating exposure and six genes were exam ined: hypoxia inducible fate 1 α , carbamoyl-phosphate synthetase 1, E3 component of pyruvate de hydrogenase, acyl-CoA de hydrogenase family member 10, and glucocorticoid receptor protei n. Of these, there was a significant decrease in gene expression of

acyl-CoA dehydrogenase (p=0.03) and glucocorti coid receptor (p=0.04) at 24 hrs. Gene expression of the remaining genes and tim e 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 at razine-treated and controls within the first 72 hrs of exposure nor in larvae tested at NF stage 62. The ADP:ATP ratio was not244 different between atrazine-t reated and control animals at 24 – 72hr samplings. The authors report a near significant increase in ADP:ATP ratios in ta dpoles exposed to 200 μg/L at NF stage 62.

The authors note that measurab le 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 atrazi ne at 200 and 400 μg/L experience an
imbalance in their energy homeostasis. Th ey conclude that based on the Langerveld et

al . 2009 study, a number of metabolic pathways were significantly ch anged in NF Stage
62 larvae exposed to 400 μg/L throughout their development a nd that there was
increased gene expression for pathways associat ed with conversion of lipids and proteins
into energy (including synthesis and degr adation of ketone bodi es, the urea cycle,
regulation of authophagy, citric acid cycle, glutathione metabolism and metabolism of a
number of amino acids. Decreased gene e xpression was associated with carbohydrate
metabolism, fat storage, protein synthesis, glycolysis/glucogenesi s, fatty acid elongation
in mitochondria and ribosomal proteins. The authors considered these changes reflective
of the changes in body weight and fat body si ze 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 in creases in gene expression a ssociated with ketone body
synthesis/degradation is an adaptive respons e to starvation at NF Stage 62 and that it
appeared that these animals depleted their glucose stores and shifted transcriptional
responses to increase fatty aci d oxidation to accom modate energy needs and that this was
consistent with increased fatty acid β oxidation. The authors acknowledge that the
increased expression of glutathi one metabolism could be a result of the fact that excretion
of atrazine is accomplished after Phas e I metabolism followed by conjugation to
glutathione and would be a ssociated the utilization (mob ilization) of amino acids.

The absence of significant effects on ATP or ADP:ATP ratios was attributed to the
successful effort by animals to respond adaptiv ely 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. Change s in body weight early in expos ure showed that atrazine245 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 expressi on reported in this article are uncertain; however, the study demonstrates up-regulation of certain ge nes and down-regulation of other genes. Given the difficulties with the Langerveld study though, the information obtained from the gene expres sion is of questionable utilit y; whereas, gene expression information from Zaya studies C and F al so 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 th e 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 re ported in surface water adjacent to field treated with atrazine . As such, the "envir onmental relevancy" of

these treatment levels is uncertain.

The article refers to Langerveld et al . 2009 to support claims that atrazine exposure at

400 μ g/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 th at so many of the test animals were not surviving to metamorphosis that the protocol had to be adju sted 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 un certain whether any meaningful relationships can be evaluated for specific chemicals within that environment.

Tadpoles were reportedly exposed from pos t-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 woul d correspond to metamorphosis when it is likely that test animals would cease feeding a nd 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 analys is samples based on the highest levels of expression. This constitutes judgment sampling and not random sampling. It is uncertain

whether differences identified in this st udy would have been apparent had random sampling been used. The authors discuss a pot ential energy imbalance as a result of atrazine exposure; however, energy balances are difficult to assess. The authors’ conclusions that the etiology of the "appa rent energy imbalance" could be due to

detoxification and excretion of atrazine and/ or endocrine disruption is broad, and with respect to endocrine di sruption, 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 explana tion for the effects they report. It is uncertain whether the study results support th e statement that "clearly, acutely and chronically exposed tadpoles are compromise d, decreasing their ability to survive the stresses of metamorphosis and/or to ade quately 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 wa y 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 uncl ear 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 larv ae; however, the compan ion article by Zaya

et al . only speculated that increased caspase-3 activity would lead to increased apoptosis in spite of the fact that no pat hology was observed thr ough liver histology.

Primary Reviewer:

Thomas Steeger, Ph.D., Senior Science A dvisor, Environmental Risk Branch 4,

Environmental Fate and Effects Di vision, Office of Pesticide Programs

Secondary Reviewer (required if study results are used quantitatively):

Not applicable247 4. References from the 2003 and 2007 SAPs on Amphibians

2003 (From Table A in the 2003 While Paper)

2007 SAP (next page)248