## APPENDIX B.4 Additional Amphibian Studies reviewed since 2012 SAP
The following studies are those that have been reviewed since the 2012 SAP and were included in the weight of evidence analysis for effects to aquatic amphibians. Previous amphibian study reviews can be found in Appendix B.2. Studies are listed below starting with the most recent date and the open literature reviews immediately follow.

Ref # Author Title Source Publicati on Year Page No.

166446 Dornelles MF;Oliveira GT Effect of Atrazine, Glyphosate and Quinclorac on Biochemical Parameters, Lipid Peroxidation and Survival in Bullfrog Tadpoles (Lithobates catesbeianus) Arch. Environ. Contam. Toxicol. 66(3): 415

- 429 2014 3

164768 Brodeur JC; Sassone A; Hermida GN; Codugnello N Environmentally

Relevant Concentrations of Atrazine Induce
Non

- Monotonic Acceleration of Developmental Rate and Increased Size at Metamorphosis in Rhinella

arenarum Tadpoles Ecotoxicol. Environ. Saf.
92: 10

- 17 2013 5 (review conducted under
EDSP program)

164775 McMahon TA;Romansic JM;Rohr JR Nonmonotonic and Monotonic Effects of Pesticides on the Pathogenic Fungus Batrachochytrium dendrobatidis in Culture and on Tadpoles Environ. Sci. Technol. 47(14): 79 58

- 7964 2013 15

161314 Paetow LJ; McLaughlin JD ; Pauli BD; Marcogliese DJ Mortality of American Bullfrog Tadpoles Lithobates catesbeianus Infected by J. Aquat. Anim. Health 25(1): 15

- 26 2013 172 Gyrodactylus jennyae and Experimentally Exposed to Batrachochytrium dendrobatidis
None Rohr JR, Raffel TR, Halstead NT, McMahon TA, Johnson SA, Boughton RK,Martin LB. Early

- life exposure to a herbicide has enduring effects on pathogen has enduring effects on pathogen induced mortality. Proc R Soc B 280
:20131502. 2013 19

165358 Rohr JR;Palmer
BD Climate Change, Multiple Stressors, and the Decline of Ectotherms Conserv. Biol. 27(4): 741

- 751 2013 23

160519 Paetow LJ;McLaughlin JD;Cue RI;Pauli BD;Marcogliese
DJ Effects of Herbicides and the Chytrid Fungus Batrachochytrium dendrobatidis on the Health of
Post

metamorphic Northern Leopard Frogs (Lithobates pipiens) Ecotoxicol. Environ. Saf.
80: 372

- 380 2012 26

160887 Svartz GV;Herkovits J;Perez

- Coll CS Sublethal Effects of Atrazine on Embryo

- Larval Development of Rhinella arenarum (Anura: Bufonidae) Ecotoxicology 21(4):
1251

- 1259 2012 293 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citati on: 166446
Dornelles M.F. and G.T. Oliveira. 2014. Effect of Atrazine, Glyphosate and Quinclorac on Biochemical Parameters, Lipid Peroxidation and Survival in Bullfrog Tadpoles ( Lithobates catesbeianus ). Arch. Environ. Contam. Toxicol. 66(3): 415

- 429.

Purpose of Review: Registration Review

Date of Assessment: 10/23/2015

Brief Summary of Study Findings: This study examined the effects of atrazine at 5, 10 and 20 μg/L on bullfrog tadpoles (Lithobates catesbeianus ) on biochemical parameters, lipid perox idation and survival. Biochemical parameters analyzed included glycogen, total lipids, triglycerides, cholesterol and total proteins in liver, gill and muscle. Atrazine exposure induced significant changes in all biochemical parameters analyzed and increas ed lipid peroxidation levels, with significant impacts to glycogen stores in all compartments. No significant changes in growth or survival in treatment groups as compared to controls were noted at the end of 14 days.

Methods : Live tadpoles were acquired from a commercial frog at 4 months of age and were in the larval

Gosner stage 26 according to Gosner (1960). A nimals were placed in 22 aquariums each containing 12 L of water (n = 12), with constant aeration, a water temperature of 22 ± 2 o C, pH 6.2 ± 0.3, and a 12

- h light

- to

- dark cycle. The tadpoles were fed (5 % of their biomass) once daily with the same fish feed used in the commercial frog farm . Animals were acclimated for 7 days with exposure occurring on day 8. Experimental design consisted of two rep licates for each exposure concentration of 5, 10 and 20 ug/L atrazine. Commercial formulation of atrazine was used ( Primo ́leo, 400 g/L; Syngenta ). The exposure period was 7 days with atrazine being applied only once in liquid formulation and dissolved in d istilled

water

. Animals were sacrificed b the freezing method in treatment groups at day 14 and in control groups at days 7 and 14. Left and right gills, liver and muscle were removed and tissue specimens were
then used for biochemical analysis of the leve ls of glycogen, total protein, lipids, cholesterol, and triglycerides as well as evaluation of lipid peroxidation levels for each treatment level and controls

.

Results : Significant differences (p<0.05) from controls were noted in the atrazine test concent rations
below :

Tissue
tested Glycogen Total protein Lipids Cholesterol Triglycerides Lipid
Peroxidation (ug/L)
Gill 5, 10, 20 None 5, 10, 20 None 5, 10 5, 10, 20
Liver 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 204 Muscle 5, 10, 20 5, 10, 20 1 5, 10, 20 5, 10, 20 5, 10, 20 5, 10, 20

1 Significant difference only noted when compared to control group sacrificed at 7 days.

No significance differences were noted in weight or survival of atrazine treated animals as compared to
controls.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment.

Rationale for Use: The study provides information on sublethal effects of atrazine on biochemical parameters as well as growth and surviva l in bullfrog tadpoles

.

Limitations of the Study: Nominal concentrations are only reported; there is no inclusion of measured concentrations in control or treatment groups.

Reported water quality parameters were dissolved oxygen, pH and temperature. No w ater renewal was noted during the 7 day treatment period. Constant aeration was reported.

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division5 

## Data Evaluation Record

ATRAZINE
PC Code 080803

EPA Contract No. EP10H001452 Task Assignment No. 4

- 4

- 2013

Study Type: Non

- Guideline

Citation: Brodeur, J.C., Sassone, A., Hermida, G.N., and Codugnello, N. (2013). Environmentally

relevant concentrations of atrazine induce non

- monotonic acceleration of developmental ra te and increased size at metamorphosis in Rhinella arenarum tadpoles. Ecotoxicology and Environmental Safety . 92:10

- 17.

Prepared for
Environmental Fate and Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency
One Potomac Yard 2777 S. Crystal Drive Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road,

B uilding 100, S uit e B Durham, NC 27713

Primary Reviewer : Sandra Hastings Signature:

Date: 11/ 25/ 2014 Secondary Reviewer : Kelly Luck, M.S. Signature:

Date: 12/3/2014 Program Manager: Jack D. Early, M.S. Signature:

Date: 12/15/2014

Disclaimer6 This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by Dynamac Corporation personnel

.7 E PA Primary Reviewer: Amy Blankinship Date: __ 7/15

___________ E PA Secondary Reviewer: Date: _____________



## Data Evaluation Record

ECOTOX NO: 164768

TXR NO : Not provided

STUDY TYPE : Non

- guideline

DP BARCODE : Not provided

PC CODE : 080803

CAS NO. : 1912

- 24

- 9

MRID NO

. : Not available

TEST MATERIAL (% purity) : Atrazine (technical grade, 98%)

CITATION : Brodeur, J.C., Sassone, A., Hermida , G.N., and Codugnello, N. (2013). Environmentally

relevant concentrations of atrazine induce non

- monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles. Ecotoxicology and E nvironmental S afety . 92:10

- 17.

SPONSOR : Not applicable (open literature review)

E XECUTIVE SUMMARY : The purpose of this study was to examine the effects of environmentally

relevant concentrations of atrazine on the timing of metamorphosis and body size at metamorphosis in
the common South American toad Rhinella arenarum

. The e xperimental design executed in the present study is a follow

- up of an earlier study ( Brodeur et al. 2009 1 ) which demonstrated a non

- monotonic
acceleration of the time to metamorphic climax (Gosner stage 42) and delayed tail resor ption

. Exposures examined in the current study we re longer (from stage 25 instead of stage 38) and the tested
concentrations we re lower and more similar to levels detected in the environment

.

Methods : Adult South America n toad s ( R

. arenarum )

, weighing approximately 200

- 250 g

, were collected from pasture fields in Buenos Aires Province, Argentina . The o vulation of female toad s was induced by an intraperitoneal injection of homologous hypoph y sis suspended in AMPHITOX solut ion

1 Brodeur

, J.C

. , Svartz, G., Perez

- C o ll, C.S.

, Marino, D

. J

. G., and Herkovits

, J

. ( 2009 )

. Comparative susceptibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis; Non

monotonous acceleration of the time to climax and delayed tail resorption

. Aquat Toxicol

. 91 : 161

- 170

.8 ( AS ; Herkovits and Perez

- Col l , 2003 2 )

. Oocytes were fertilized in vitro using fresh sperm suspended in

AS , and t he resulting embr yos were maintained in AS at 20± 2 ° C until reaching stage 25. W hen tadpoles began feeding at stage 24

- 25 , they were offered boiled Swiss chard ad libitum

.

Tadpoles were pooled from four different clutches , and t wenty replicates were performed for e ach
concentration tested

. In e ach replicate, ten tadpoles (at stage 25 ) were placed in AS with or without
test substance

. Tadpoles were exposed to atrazine (98% purity, Chem Service, West Chester, PA) at 0.1

, 1

, 10, 100 or 1000 μ g/L . A stock solution of atrazine (10 mg/L) was prepared in AS using acetone as a solvent , and the test solutions were prepared by sequentially dil uting the stock solution with AS; the atrazine concentration in the stock solution was verified by LC/MS/MS analysis. Additionally, the study included a control group exposed to AS only ; a solvent control group expose d to AS containing 0.02% (v/v) acetone ; and a positive control group exposed to 100 μ g/L of 17 β

- estradiol (E2 ; prepared in AS from a stock solution in acetone)

. To avoid evaporation of test solution, experimental tanks consisted of two superposed glass Petri dishes

. Test solutions were replaced entirely every 48 h, and the temperature was maintained at 20±2 ° C throughout the experiment. Dead tadpoles were removed and
the G osner stage and survival were evaluated every other day when renewing the solutions

. Following 40 days of exposure, tadpoles were transferred to two bigger superposed glass Petri dishes containing test solution to provide space to the growing tadpoles. Frogs completing metamorphosis were
examined for malformations

, and their body mass and snout

- vent length (SVL) were measured

.

The study authors used a linear regression of body mass on SVL to evaluate body condition

. D ata from
the control groups were used to perform th is regression analysis. The theoretical body weight value of frogs from both control and treated gro ups (obtained by introducing the length of the animal in the equation of the regression line) was then subtracted from the measured body weights to obtain a

“ residual" value

, represent ing an expression of the difference between the measured weight of the

m etamorph and that of an average healthy subject w ith the same SVL. A n indiv idual with a positive
residual wa s considered to be in a good condition whereas an individual with a negative residual wa s regarded as having a reduced body condition.

Comparisons between the control and solvent control groups were made using a t

- test or a rank sum

test if normality and equal variance could not be obtained

. Because there were no differences between
the two control groups

, data from both groups were comb ined for further analyses . F our

- parameter logistic regression analysis wa s conducted for each treatment on the cumulative numbers of animals reaching stage 39, stage 42 or completing metamorphosis as a function of exposure duration (G raphPad
Prism softwar e )

. For each curve, the time for 50 % of the individuals to reach the stage being examined,

s tage 39 (T39), stage 42 (T42) or completion of metamorphosis (TCM)

, was calculate d. The duration of
the intervals between stage 39 and stage 42

, and between stage 42 and completion of metamorphosis were calculated by subtracting the values ( the value of T39 from the value of T42

, or the value of T42
from the value of TCM ) for each replicate of e ach treatment

. The values of T 39, T 42 and TCM, the SVL s of the metamorphs, and the intervals between s tage 42 and completion of metamorphosis were

2 Herkovits , J. and Perez

- Col l

, C

. S. ( 2003 ) AMPHITOX: a customized set of toxicity tests employing amphibian embryos

. In: Linder G.

, Sparling, D.W.

, Krest

, S., Little, E . (Eds .), Multiple Stressor Effects in Relation to Declining Amphibian Populations

, American Society for Testing and Materials International

, Bridgeport , NJ, USA

. pp. 46

- 60

.9 compared between treatments using o n e

- way analysis of v ariance (ANOVA) followed by a Holm

- Sidak test for multiple comparisons

. A non

- parametric Kruska l

- Wall is one

- way ANOV A on Ranks was used to compare the following parameters for which normality and equal variance could not be obtained :

s urvival of tadpole s; intervals between stage 39 and stage 42 ; proportions of individuals reaching stage 39, stage 42 or completing metam orphos is; body mass of metamorphs ; and residuals describing body
condition . All t

- tests
, ANOVAs

, and multiple comparison tests were conducted using SigmaStat 3.11
statistical software . Significance was determined at p<0.05.

Results : T he only concentration of atrazine that had significant mortality was 1000 μ g/L

, at which
survival was reduced to 52

. 0 ± 10.0 %, compared to 74.2 ± 6.5% for the control group and 78.0 ± 8.5% for the E2 group. Survival rates in the 0.1, 1, 10, and 100 μ g/L treatment groups (75.7

- 82.5%) were not significantly different from the control or E2 groups. In all atrazine treatment groups, most of the mortalities occurred during the first 20 days of exposure (data not reported).

T he progression of the metamorphosing tadpol es to stage 39, stage 42

, and completion of metamorphosis is presented in Fig ures 1 , 2, and 3 , respectively (copied without alteration from Brodeur et al. 2013), along with the time required for 50% of the tadpoles to reach that stage for each treatment

gr oup

. Tadpoles exposed to atrazine at 1000 μ g /L took significantly (p<0.05) more time than controls to reach stage 39 (increased T39)

, whereas expos ure to 1, 10 and 100 μg/L significantly reduced T39 (Fig ure 1b) compared to the controls. Th is acceleration of development with atrazine exposure had a U

shaped concentration

- response relationship as 10 μ g /L caus ed a greater reduction of T 39 than 1 or 100

μ g/L (which generat ed comparable effect s). A simila r U

- shaped pattern of acceleration of development

as a function of at r azine concentration was observed when considering T42 (Fig ure 2b) and TCM (Fig ure 3b).

Tadpoles exposed to 17

- estradiol (100 μg/L) showed acceleration of development of the same amplitude and characteristics as those observed in the atraz ine 1 and 100 μg/L treatment groups (Figures 1

- 3).10 Figure 1

. (a) Cumulative number of individuals reaching stage 39 over time for control larvae of Rhinella arenarum and larvae exposed to atrazine and 17 β

- estradiol. The lines represent the sigmoidal curve models calculated for

every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to reach stage 39 for controls and larvae exposed to atrazine and estradiol. Controls are denoted by C. The number indicated at the
side of the abbreviation ''At" represents the concentration of atrazine in μ g / L

. E2=tadpoles exposed to 100 μ g /L of

17 β

- estradiol

. Bars with the same letter are not significantly different (p<0.05)

.

Figure 2

. (a) Cumulative number of individuals re aching stage 42 over time for control larvae of Rhinella arenarum and larvae exposed to atrazine and 17 β

- estradiol. The lines represent the sigmoidal curve models calculated for

every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to reach stage 42 for controls and larvae exposed to atrazine and estradiol. Controls are denoted by C. The number indicated at the
side of the abbreviation ''At" represents the concentration of atrazine in μ g / L

. E2=tadpoles exposed to 10 0 μ g /L of

17 β

- estradiol

. Bars with the same letter are not significantly different (p<0.05)

.11 Figure 3

. (a) Cumulative number of individuals c ompleting metamorphosis over time for control larvae of Rhinella

arenarum and larvae exposed to atrazine and 17 β

- estradiol. The lines represent the sigmoidal curve models
calculated for every treatment (b) Time required (mean ± SE) for 50 percent of the larvae of Rhinella arenarum to complete metamorphosis for controls and larvae exposed to atrazine and estradio l. Controls are denoted by C. The
number indicated at the side of the abbreviation ''At" represents the concentration of atrazine in μ g / L

. E2=tadpoles exposed to 100 μ g /L of 17 β

- estradiol

. Bars with the same letter are not significantly different (p<0.05)

.

T he duration of the interval between stage 39 and stage 42, and between stage 42 and completion of metamorphosis are presented in Fig ure 4 (copied without alteration from Brodeur et al. 2013 ) . The interval between stages 39 and 42 was significantly decreased at 1000 μg/L compared to controls.
There we re no significant differences between treatment groups and controls for the interval between stage 42 and completion of metamorphosis. The stu dy authors concluded that the impact of atrazine on metamorphosis occurred entirely between stage 25 and stage 39 for atrazine concentrations between 1
and 100 μ g/L

.

It was noted that t he effects on metamorphosis in the 1000 μg/L group differed from eff ects caused by
atrazine exposure at lower concentrations. The initial significant slowing of development observed between stage s 25 and 39 (Figure 1b) was followed by a significant decrease in the duration of the interval between stage 39 and stage 42 (Fi gure 4)

, which caused the overall duration of metamorphosis
to be similar to controls (Figure 3b)

.

The only tested concentration of atrazine that did not affect the timing of metamorphosis was 0.1 μg/L ; therefore, it was concluded that this concentratio n represented the no observed effect concentration (NOEC).12 Figure 4

. (a) Duration of the interval (mean ± SE) between when 50% of Rhine lla arenaru m larvae reach stage 39 and when 50% reach stage 42. (b) Duration of the interval (mean ± SE) between when 50% of Rhinella arenarum larvae reach stage 42 and when 50% complete metamorphosis

. Controls are denoted by C. The number indicated
at the side of the abbreviation ''At" represents the concentration of atrazine in μ g / L

. E2 =tadpoles exposed to 100

μ g /L of 17 β

- estradiol . * = significantly different from controls (p<0.05)

.

Body mass and SVL at completion of metamorphosis for control and exposed tadpoles are presented in Fig ure 5 (copied without alteration from Brodeur et al. 2013)

. Tadpoles exposed to 17 β

- estradiol or atrazine at 1 or 10 μ g/ L grew into significantly (p<0.05) longer and heavier metamorphs than controls

. A
similar result was obtained when body size data were examined in terms of body condition ( Fig ure 6

, copied without alteration from Brodeur et al. 2013 ); for these data, a significant increase was also found
f o r tadpoles exposed to atrazine at 100 μ g/L . T he concentration response curve t ook the form of an
inverted U in this case. It was concluded that t he NOEC f o r size and condition at metamorphosis was 0.1

μ g/L

.

Figure 5

. (a) Body mass at completion of metamorphosis (mean ± SE) for control larvae of Rhine ll a arenarum and larvae exposed to atrazine and 17 β

- estradiol. (b) Snout

- vent length at completion of metamorphosis (mean ± SE)
for control larvae of Rhinella arenarum and larvae exposed to atrazine and 17 β

- estradiol

. Controls are denoted by
C. The number indicated at the side of the abbreviation "At" represents the concentration of atrazine in μ g/L. E2 = tadpoles exposed to 100 μ g/ L of 17 β

- estradiol

. * = significantly different from controls ( p <0.05)

.13 Figure 6

. (a) Distribution of body mass in function of snout

- vent length for recently metamorphosed control larvae
of Rhinella arenarum and larvae exposed to atrazine and 17 β

- estradiol . T he regression line describes the relationship observed between body mass and snout

- vent length in control animals. (b) Residual (mean ± SE) between measured body mass and the theoretical body mas s of a control animal calculated by applying the equation of the regression line presented above (a) to snout

- vent length data of animals exposed to atrazine and

17 β

- estradiol

. Controls are denoted by C. The number indicated at the side of the abbreviatio n "At" represents the
concentration of atrazine in μ g/L. E2 = tadpoles exposed to 100 μ g/ L of 17 β

- estradiol

. * = significantly different from controls ( p <0.05)

.

Conclusions : The study authors concluded that l ow atrazine concentrations in the range of 1

- 100 μ g/ L
were found to accelerate the developmental rate in a non

- monotonic U

- shaped concentration

- response relationship , and that t his observed acceleration of the metamorphic process occurred entirely between stages 25 and 39 ( treated tadpoles proceed ed through metamorphosis similar to control animals beyond this point ). Furthermore, tadpoles exposed to a tr azine concentrations in the range of 1

- 100

μ g/L transformed into significantly larger metamorphs than controls

, the concentration

- response curve taki ng the form of an inverted U in this case

. The NOEC was 0.1 μ g /L atrazine for both size at metamorphosis and timing of metamorphosis. Tadpoles exposed to 100 μ g/ L 17 β

- estradiol presented the exact same alterations of developmental rate and body size as t hose exposed to 1

, 10 and 100 μ g/ L
of atrazine.

The study was conducted in Buenos Aires

. Argentina at the I nstituto de Re cursos Bio l ó gicos, Centro
Naciona l de lnvestiga ci ones Agrope cuarias , lnstituto Nacional de Tecnolog í a Agrope cuaria; the Amphibian Bi ology La boratory

- Animal Histology Biodiversity and Experimental Biology Department

, Fa cu lty of Exact Sciences, University of Buenos Aires ; and the Ecology Laboratory

, University National of

L ujan . The Good Laboratory Practices status of this research is unknown.

The study is of sufficient quality to include qualitatively in the weight of evidence for EDSP. However, the following study limitations are noted:



### • The adult frogs used to generate tadpoles for this study were caught in the wild (pastures in Bu enos Aires); any potential previous pesticide exposure is not known.

• The source of the fresh sperm used to fertilize the oocytes was not specifically stated, although it is assumed that the wild

- caught adult male frogs were the source of the

sperm.14 

### • Individ ual data for mortalities in each group were not reported.

• No quantitative data were provided for the study results; all results were presented in bar graphs.15 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: 164775 McMahon TA; Romansic JM; Rohr JR. 2013. Nonmonotonic and Monotonic Effects of Pesticides on the Pathogenic Fungus Batrachochytrium dendrobatidis in Culture and on Tadpoles. Environ. Sci. Technol. 47(14): 7958

- 7964.

Purpose of Review: Registration Review

Date of Assessment: 4/5/2015

Brief Summary of Study Findings: This study examined the effects of atrazine and chlorothalonil on the
survival of Bd both in culture and on Osteopilus septentrionalis (Cuban treefrog) tadpoles. In the concentration range tested ( 0.011 to 212 μg/L ) atrazine reduced levels of Bd growth in culture and
reduced Bd growth on tadpoles as compared to controls. Overall tadpole survival was impacted by Bd but not atrazine exposure. The atrazine treated tad poles had significant decreases in the time to death as compared to controls and increased mass of tadpoles as compar ed to the chlorothalonil group.

Methods: Bd culture was prepared using Bd stock isolated from Rana Catesbenia from Savanah River site, SC and culturing in tryptone broth. Zoospores were grown on agar plates and used for experimental
inoculation.

Effects of the two pesticides on Bd growth in culture were measured after one and two weeks.
Chlorothalonil was tes ted at 0.0176, 0.176, 1.76, and 17.6 μg/L and atrazine at 1.06, 10.6, 58, and 106 μg/L; both pesticides we re technical grade, purity >98% ( Chemservice, West Chester, PA) and two
controls were prepared for each pesticide (water and solvent ; acetone). Actual concentrations of the stock solutions were measured at 176 ug/L chlorothalonil and 106 ug/L atrazine. Culture tubes were
incubated at 23 °C for either one or two weeks and Bd concentrations were quantified at the one and
two week time increments based on 20

- uL aliquots on a hemocytometer.

Effects of the two pesticides on Bd growth in culture were also measured based on daily Bd growth for 8 days and 10 treatments based on 10

- fold serial dilutions of the pesticide ( chlorothalonil: 0.000176, 0.00176, 0.0176

, 0.176, 1.76, 8.8, 17.6, 35.2, 90.2, and 176 μg/L; and atrazine: 0.011, 0.106, 1.06, 10.6, 23, 58, 77, 106, 159, and 212 μg/L) . Effects were determined by measuring changes in Bd density using spectrophotometric analysis.

Effects of the two pesticides o n Bd growth in Osteopilus septentrionalis (Cuban treefrog) tadpoles were also determined based on four concentrations of technical grade chemical ( chlorothalonil: 0.0176,0.176, 1.76, and 17.6 μg/L; and atrazine: 1.06, 10.6, 58, and 10616 μg/L). T adpoles were collected from three clutches naturally oviposited in outdoor wading pools and were completely mixed before being distributed among treatment groups . All tadpoles were Gosner stage 25 with an average tadpole weight at the start of the experiment of 0.086 ± 0.01 g (mean± SEM
based on 10 randomly selected tadpoles). Tadpoles were housed in 500 mL glass mason jars and kept on

a 14:10 light/dark cycle . Water was exchanged weekly and Bd and pesticide treatments re

- dosed after each water change. Bd exposure was attained through inoculation with 3 mL of Bd + inoculum (concentration of Bd + was 6.0 x 10 4 zoospores/mL). Bd was quantified based on PCR analysis of tadpole mouthparts.

Results : Atrazine exposure resulted in a monotonic negative impact on Bd growth in culture, but only the highest concentration tested 17.6 ug/L significantly reduced concentrations compared to controls. For daily Bd growth as measured using spectrophotometric analysis after correcting for pesticide effect on absorbance, all at razine concentrations significantly reduced Bd growth as compared to controls except for the 0.1 ug/l concentration. For Bd growth on tadpoles, atrazine exposure resulted in significantly less Bd growth at all concentrations as compared to controls. The au thor also reported that

t adpoles exposed to atrazine died sooner than control animals or those exposed to chlorothalonil (512.4 ±11.18, 533.8 ± 10.06, 534.5 ± 10.38 h, respectively; F 1, 136 =4.98, p = 0.03 ) and higher mass than tadpoles exposed to chloroth alonil treatments (mean ± SEM: 0.26 ±0.02 g and 0.21 ± 0.01 g, respectively; F 1, 129 = 8.21, p = 0.005).

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment.

Rationale for Use: The study pro vides information on effects of atrazine on Bd growth and some limited information on tadpole growth and time to death after atrazine exposure.

Limitations of the Study: Regarding the effect of atrazine on survival time (tadpoles died sooner) and increased mass, no supporting raw data was reported to support this analysis and the effect is not
associated with a definitive concentration.

Water quality parameters not reported in study.

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmenta l Fate and Effects Division17 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: 161314 Paetow LJ; McLaughlin JD; Pauli BD; Marcogliese DJ

. 2013. Mortality of American Bullfrog Tadpoles

L ithobates catesbeianus Infected by Gyrodactylus jennyae and Experimentally Exposed to Batrachochytrium dendrobatidis

. J. Aquat. Anim. Health 25(1): 15

- 26

.

Purpose of Review: Registration Review

Date of Assessment: 4/ 6 /2015

Brief Summary of Study Findings: This study investigated the effects of atrazine on larval American bullfrogs ( Lithobates catesbeianus ) experimentally exposed to Bd. After the start of the experiment, the sample population was found to be infected with gyr odactylus jennyae leading to mild to severe skin erosions in the experimental population. Increased mortality was observed between control groups and
Bd and Bd + atrazine groups, but no difference was found between Bd and Bd + atrazine groups. The
relations hip between the co

- infections and the severity of skin lesions was correlated with their
influence on survival.

Methods : 102 bullfrog tadpoles ( (Gosner stages 26

– 37) were obtained from an Environment Canada laboratory located in Moncton, New Brunswick ( originally purchased from a commercial source in the United States). A virulent strain of Bd was used (JEL423) provided by J.E. Longcore (University of Maine, Orono). Tadpoles were randomly selected for each treatment group.

Four experimental treatment and control groups were established: (1) a group of tadpoles acutely exposed to two doses of Bd zoospores (2) a group exposed for 14 d to atrazine, followed by two acute
doses of zoospores (3) a group exposed for 14 d to glyphosate, followed by two acute d oses of zoospores and (4) a group that received no experimental exposures. There were four replicates for each group, except for the control group, which had five; each replicate consisted of six tadpoles. Nominal chemical exposure concentrations were 21 μ g of atrazine, and 1.5 mg acid equivalents (a.e.) of glyphosate, per liter of water. In the herbicide exposed animals, the two acute exposures to Bd occurred

at 14 and 16 d after the start of the pesticide exposures, and each Bd exposure lasted 24 h

.

Each replicate group was maintained in a 33

- L aquarium containing 8 L of aerated, dechlorinated water.
Three

- quarters of the water in each tank was changed every 3 d. During the pesticide exposure phase, the herbicides were replenished with each water change. Water volume was maintained at 1.3 L per tadpole and reduced by 1 L with each tadpole’s death, to a minimum of 4 L. At day 45, all surviving tadpoles were killed by immersion in a 0.8% solution of MS

- 222 (tricaine methanesulfonate). Tadpoles that died or w ere killed were immediately fixed in Bouin’s18 solution, in a 70% solution of ethanol, or in a 10% solution of buffered formalin (two tadpoles in each solution per aquarium).

At day 0, a stock solution of each herbicide was prepared. During the pesticide exposure phase of the study, each liter of water that was siphoned from the exposure tanks every third day was replaced with

1 mL of pesticide stock solution

. At two time points (at 12 h after herbicide addition and at day 14), water samples were drawn dir ectly from two randomly selected pesticide

- containing aquariums (one containing atrazine and one containing glyphosate) to compare the actual and nominal concentrations.

The concentrations of glyphosate measured at 12 h and at 14 d post addition of the pe sticides to the aquariums were both 2.1 mg/L a.e., which was 1.4 times higher than the nominal concentration of 1.5 mg/L a.e. The concentrations of atrazine measured on the same days were 140 μg/L and 77 μg/L, respectively, which were 6.7 times and 3.7 tim es higher than the nominal concentration of 21 μg/L.

Results: By day 30 of the experiment, a consistent pattern of dermal depigmentation was observed around the eyes and at the base of the tail of tadpoles in every group, including the control group

. At days 30 and 31, two tadpoles that displayed severe depigmentation (each from the group exposed to
both glyphosate and Bd ) were removed from the experiment for examination. Depigmentation was
found to be the result of skin erosion caused by the tadpoles’ in fection by the monogenean ectoparasite G. jennyae. Further examination revealed that G. jennyae was present in each aquarium.

Increased mortality was observed between control groups and Bd and Bd + atrazine groups, but no
difference was found between Bd and Bd + atrazine groups. Increased susceptibility to Bd due to atrazine exposure was not demonstrated in the study.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment , but with limited uti lity

.

Rationale for Use: The study provides information on effects of Bd on survival and the effects of co

infection of tadpoles with monogenean ectoparasite G. jennyae . However, effects due to atrazine alone
could not be discerned from those due to Bd a lone

.

Limitations of the Study: The presence of the co

- infection in all experimental animals with the

m onogenean ectoparasite G. jennyae confounded interpretation of results.

There is uncertainty in the concentrations of atrazine based on the measured concentrations (14 ug/L and 77 ug/L) as compared to the nominal concentration of 21 ug/L.

Water quality parameters not reported in study.

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division19 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: 165358

Rohr J

. R . and B.D. Palmer. 2013. Climate Change, Multiple Stressors, and the Decline of Ectotherms

. Conserv ation Biol ogy. 27(4): 741

- 751

.

Purpose of Review: Registration Review

Date of Assessment: 10/23/2015

Brief Summary of Study Findings:

This study examined the effects of temperature (22oC or 27 oC), moisture (wet or dry) and atrazine (0, 4, 40 and 400 μg/L) on the survival, growth, behavior and foraging of streamside salamanders ( Ambystoma
barbouri ). Changes in temperature and moisture alone caused significant loss of mass and mortality with atrazine exposure having an add itive effect by decreasing water conserving behavior, foraging efficiency, mass and time to death. Compared to controls, at the end of the experiment there was a significant negative effect on the percent of mass change at all atrazine concentrations teste d as well as a negative association between atrazine concentration and time of survival. Temperature had the greatest impact on individual fitness, with higher temperatures having a higher impact on survival than moisture.

Methods : Streamside salamanders were exposed to atrazine during their larval stage from the embryo stage (April 2002) to metamorphosis ( August 2002). No further atrazine exposure occurred after metamorphosis. Methods were briefly summarized in the study and the rea der is referred to Rohr 2004 for further details. 31 eggs were placed into 48 aquaria containing 9.5 L at pH of 8 and temperature of
15◦C. Egg clutches were collected from Fossil Creek (Jessamine County, KY). Four atrazine concentrations, each containing solvent DMSO, were tested at 0, 4, 40, and 400 ppb (80% pure atrazine). Water was changed weekly and the chemical reapplied with weekly measurements of atrazine taken and reported as 4.95 μg/L (SE 0.30), 38.70 μg/L ( SE 2.14), and 386.04 μg/L ( SE 23.24)

.

For the 11 week post metamorphosis observation period 160 terraria were constructed. 3 metamorphs from each treatment group were placed in a terrarium. Two incubators were established at 22◦C and 27◦C. The experimental layout consisted of:20 Atrazine aq uatic exposure
concentration prior to metamorphosis (ug/L) Terrestrial temperature/moisture exposure matrix
Incubator 1:

22 ◦ C Incubator 2:

22 ◦ C Incubator 3:

2 7◦C Incubator 4:

2 7◦C

Wet Dry Wet Dry Wet Dry Wet Dry

0

4

40

400

1 dry = 42% moisture by weight, wet = 78% moisture by weight

Temperature selection was based on the natural conditions of salamand ers in Kentucky (22 ◦ C) with the

27◦ C temperature intended to be both representative of a possible range of temperatures associated with global warming but also within the bounds of current natural c onditions (Kentucky temperatures
reach 30.5 ◦ C in summer). Cool and moist conditions were anticipated to provide the most favorable conditions and warm and dry conditions expected to be associated with the most significant adverse
effects.

Each terrarium w as provided one cricket per salamander per week. Individual salamanders were denoted by toe clipping. Thirteen times (approximately once weekly) during the procedure observations
were made if salamanders were in refuge (under a refuge plate or buried), mov ing, huddled, or climbing up the walls of the terrarium . Mass and length were measured during the last week of the experiment. Survival was assessed once weekly when the terraria were weighed as it sometimes required disturbing buried salamanders. Foraging trials were conducted during the last week of the experiment where 4 crickets were placed in each terraria and number of crickets eaten and number of salamanders buried was recorded hourly for 4 hours. This was only conducted in the 22◦C incubators due to high mortality at this point in the experiment in the 27◦C terraria.

Results : From the summary of results, the following interactions were noted:

Endpoint measured NOAEC (ug/L) LOAEC (ug/L)
Behavior

– Huddling 4 40
Behavior

– Inactivity <4 4
Behavior

– In refuge 4 40 Mass change <4 4*
Survival >400 NA

*p=0.054 at 40 ug/L atrazine Each square represents separate
incubator conditions that contain 5 replicates (terrariums) with 3 metamorphs in each replicate21 Significant effect was noted on certain behaviors at all atrazine concentrations; however, as presented, results were pooled from all temperature and moisture test conditions together making it difficult to separate out effects due to atrazine alone. Signi ficant differences on behavior were also reported due to temperature and moisture changes when all atrazine exposure concentrations pooled. A significant effect on mass change was noted with atrazine exposure overall. A 4% change with each log 10
differen ce was noted at 27oC.

In foraging trials (performed only on 22oC incubators due to high mortality in 27◦C incubator groups), 1 hr after exposure most salamanders were not buried in wet or dry conditions. Under dry conditions, 1 hr
after release of cricke ts, atrazine exposed salamanders had reduced foraging efficiency as evidenced by the number of crickets eaten per salamander. However, due to the effect of atrazine to reduce the number of salamanders in refuge under dry conditions, at 2

- 4 hrs there was an increase in foraging efficiency as less salamanders were buried by 4 hrs. Under wet conditions, atrazine exposure was associated with a negative effect in foraging efficiency at all time points. The author noted the
cumulative number of crickets eaten de pended significantly on time, moisture and atrazine exposure.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment.

Rationale for Use: The study pro vides information on amphibian growth, mo rtality and behaviors under
different and changing environmental conditions; implications for multiple stressors in the environment;
uncertainties identified are described below.

Limitations of the Study: DMSO was used as the solvent without a negative co ntrol. Uncertainty exists in the use of DMSO as a solvent as it may accelerate the movement of chemicals across cell membranes. However, the author reported that no negative control was included as the solvent has not been shown
to have effects on behavior , growth or survival in this species in a previous study (Rohr et al., 2003a).

All results were only presented graphically, making it difficult to validate or further analyze the reported results. In addition, for certain endpoints, it was difficult to discern if the reported effects associated with atrazine exposure were from pooled results of all temperature and exposure concentrations or individual exposure regimens. As significant effects were seen due to changes in temperature and moisture alone, th is made it challenging to differentiate a clear causation from atrazine exposure alone.

The aqueous phase exposure portion of this study was previously reported under Rohr 2004. The 2004 study was previously reviewed (Open literature summary review, A. Pease and A. Blankenship, 2012). Concern was noted in the study review due to a possible fugal pathogen forming a white film on embryos, use of DMSO, no negative control, no raw data reported, unknown study duration, variation in results from 2002 to 2003, possible prior exposure of test organisms to atrazine, high loading rates and the lack of ammonia and nitrate reporting.

R eviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division22 References:

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

- Carthy, J. H. Niedzwiecki, T. Sager, A. Sih, and B. D. Palmer.2003a. Lethal and sublethal effects of atrazine, carbaryl, endosulfan, and octylphenol on the streamside salamander, Ambystoma barbouri. E nvironmental Toxicolog y and Chemistry 22:2385

– 2392.

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. 2004. Multiple stressors and salamanders: effects of an herbicide, food limitation, and hydroper iod. Ecological Applications 14:1028

– 1040.23 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: None Rohr JR, Raffel TR, Halstead NT, McMahon TA, Johnson SA, Boughton RK,Martin LB. 2013. Early

- life exposure to a herbicide has enduring effects on pathogen induced mortality. Proc R Soc B 280
:20131502.

Purpose of Review: Registration Review

Date of Assessment: 4/ 6 /2015

Brief Summary of Study Findings : This study examined t he effects of 6 day atrazine exposure at 65.9 μg/L to Cuban tree frog tadpoles ( Osteopilus septentrionalis ) on the response to Bd exposure both immediately after atrazine exposure as tadpoles and 46 days later (post

- metamorphosis). Tadpoles were exposed to at razine during the first week (window 1; Gosner stage 26

- 28) and the second week (window 2; Gosner stage 35

- 37) of development. A significant difference was found in tadpoles exposed during window 2 but not window 1 in SVL and time to metamorphosis; those t adpoles exposed during window 2 were smaller and morphed earlier than window 1. Mortality was significantly higher in atrazine exposed groups when exposed to Bd both immediately and 46 days later (post metamorphosis) as compared to solvent controls exposed to Bd

.

Methods: 60 outdoor mesocosms (1.8 m diam eter, 60 cm deep, approx. 1100 L ) were established, each containing 800 l of water and inoculations of local zooplankton, phytoplankton and periphyton, and
covered with 60% shade cloth. Forty tanks received 50 O. septentrionalis tadpoles (all at Gosner stages [25

– 28 ]) from five clutches collected from pools at the University of South Florida (USF) campus (the clutches were mixed to homogenize genetic variation before being distribu ted among the tanks). P ools
were filled with municipal water and could not receive any run

- off that might contain contaminants. This experiment was conducted in outdoor tanks, so that the food resources of the tadpoles might be more
natural than in a labor atory.

The study uses a completely randomized 2 x 2 x 2 x 2 factorial design with five replicates of each test group. Tanks received either 25 ml of ethanol solvent or atrazine (Chemservice, West Chester, PA;technical grade, purity more than 98% ) dissolved in 2 5 ml of ethanol. A water control was not included due to previous studies by the author that never detected any immediate or later

- life effects of the solvent on any measured traits. W ate r samples were taken from every tank 1 h after dosing and atrazine wa s quantified using the Abraxis (Abraxis LLC,Warminster, PA) ELISA microtiter plate kit, with each sample processed in duplicate (measured c oncentrations were 65.9+3.48 ug/L 3 , mean+s.e.).

3 This concentration was erroneously reported as 65.9 mg/L in the originally manuscript but is corrected to 65.9 ug/L in a correction issued to the article.24 T adpoles were exposed to the solvent control or atrazine for 6 days at one of two periods during their development, either in the first week (Gosner stage 26

– 28) or second week (Gosner stage 35

– 37) of the experiment. After exposure to atrazine or solvent, Gosner stage and snout

– vent lengths (SVLs) of all tadpoles was characterized and tadpoles were exposed to Bd or not . To test for short

- and long

- term effects of atrazine exposure on disease risk, the pathogen treatments occurred less than 18 h or a mean of 46 days after the end of the chemical exposure period. Th e latter Bd exposure time was post

metamorphosis. If a tank of tadpoles was designated to be exposed to the Bd treatment post metamorphosis, then all the tadpoles from the tank were counted and transferred to an adjacent tank
after the 6

- day atrazine

- or s olvent

- exposure period and then reared to metamorphosis. T hese adjacent tanks were established the same way as the ‘exposure’ tanks but were free of atrazine or solvent. Metamorphs were pulled from the tanks every other day and maintained in the laboratory (12 h light cycle, 238C) on vitamin

- and mineral

- dusted crickets (fed ad libitum) until their use in the Bd

- exposure portion of the experiment. In summary, there were 60 mesocosms total; 40 served as the primary tanks
for the 2x2x2 portion of the experime nt (atrazine or solvent x early or late in development x immediate or delayed Bd exposure), and 20 mesocosms had freshwater to house the delayed Bd exposure animals
in the absence of atrazine or solvent.

Tadpoles were exposed to 2 ml of the Bd + or Bd

- (pr epared identically to Bd + but lacked Bd zoospores)
inocula in 80 ml of artificial spring water for 2 days

. Metamorphic frogs were exposed to the Bd by pipetting 2 ml of 3 x 10

- 4 zoospores ml

- 1 onto the frog’s ventral side. In general, five tadpoles or

meta morphs were exposed to Bd from each tank, but these numbers varied depending on survival. Post

- metamorphic frogs were fed five vitamin

- and mineral

- dusted crickets (5

– 15 mm) twice per week, and tadpoles were fed organic spinach ad libitum. During the Bd + or Bd

- exposure periods, tadpoles and metamorphs were maintained at 23 ◦ C on a 12 h light cycle and their
survival was monitored daily for 14 days. PCR analysis was used to quantify Bd from tadpole mouthp arts (dissected) and hindlimbs.

Results: No factor s had significant effects on tadpole survival during the 6

- day atrazine or solvent exposure. Atrazine had no significant effects on survival to metamorphosis in the absence of Bd nor any interactions of other factors with atrazine were significant predicto rs of mortality. Atrazine exposure early in life significantly increased mortality in the presence of Bd indicating a significant interaction
between Bd and earlier exposure to atrazine. Risk of death was two times more likely for individuals exposed to at razine and B d and atrazine then those exposed to solvent and Bd . There was a similar magnitude of mortality when frogs were exposed to Bd immediately after the 6

- day exposure to atrazine or later in life (mean = 46 days ), suggesting there was no evidence o f recovery from early

- life exposure to atrazine. The authors provide discussion on factors possibly contributing to the observed results, including the impact of atrazine exposed individuals being of reduced size and accelerated development at the time of Bd exposure and also the possible direct impact of atrazine on the endocrine and immune systems. The latter theory was further investigated by controlling for development and SVL and still demonstrating increased Bd

- mortality in atrazine exposed individuals.

D espite atrazine

- exposed frogs having significantly greater Bd

- induced mortality than solvent

- exposed frogs, there was no evidence that the atrazine

- exposed frogs were less resistant to Bd.25 T olerance differed significan tly among the atrazine

- and solvent

- exposed frogs; at the same Bd load,
atrazine

- exposed frogs were more likely to die than solvent exposed frogs.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

ass essment.

Rationale for Use: The study provides information on the impacts of atrazine exposure in early life stages to mortality from Bd infection

.

Limitations of the Study: Although tanks started with 50 tadpoles each, it is unclear the number of frogs that were ultimately exposed to Bd for each group, although it was noted generally 5 frogs from each tank were exposed. Summary statistics but no raw data were provided.

Muni cipal water was used to fill tanks; water quality data was not reported.

Nominal concentrations not reported; atrazine concentrations only monitored on Day 1 (1 hour post treatment).

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division26 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: 160519
Paetow LJ

, McLaughlin JD

, Cue RI , Pauli BD, and DJ Marcogliese. 2012. Effects of Herbicides and the Chytrid Fu ngus Batrachochytrium dendrobatidis on the Health of Post

- metamorphic Northern Leopard Frogs ( Lithobates pipiens )

. Ecotoxicol. Environ. Saf. 80: 372

- 380

.

Purpose of Review: Registration Review

Date of Assessment: 11/4 /2015

Brief Summary of Study Findings : The effects of atrazine and the chytrid fungus Batrachochytrium
dendrobatidi (Bd) were investigated. Post

- metamorphic northern leopard frogs ( Lithobates pipiens ) were exposed to atrazine at 2.1 μg/L (actual levels 1.70 to 4.28 μg/L) alone for 21 days then followed by
Bd and observed for 94 days. Significant effects on mass gain were seen in the atrazine treated group over the course of the experiment (Days 1

- 94) with 11% and 8% decrease in mass gain as compared to
control means in atrazine alone a nd atrazine + Bd groups collectively. No significant effects were seen on survival, biomarkers of animal health or immune function. There was no evidence of active Bd infection in the frogs at the end of the exposure period, which the author attributed to a potentially resistant strain of frogs and significantly increased skin shedding in exposed frogs which may have helped resist or clear infection.

Methods : Recently metamorphed northern leopard frogs were captured from a wildlife reserve near Boucherville, Quebec. Water samples were taken from the pond the frogs were captured from and analyzed for 39 pesticides, including atrazine. Twenty random individuals were immediately swab bed to test for Bd infection. During the 8 week acclimation period, frogs were housed in individual 1.84 L plastic containers containing 50 mL of dechlorinated, UV

- treated water and two platforms permitted frogs to
exit the water. Acclimation included a 16 h:8 h light:dark photoperiod and room temperature of 21 ±1.3

◦ C. Frogs were measured and weighed prior to the start of the experiment. Sixty

- four frogs were exposed to atrazine at 2.1 ug/L (Aatrex © Liquid 480, Syngenta Crop Protection Canada, Inc.) for 21 days using 1 L mason jars containing 50 mL of treatment solution. Jars were held at a slant (except days of Bd exposure) to allow frogs to enter and leave the liquid and mimic a semi

- terrestrial environment. Water was changed daily during the acclimation and exposure period. Pesticide was diluted from stock solution to achieve the desires test concentrations. Diluted stock solution on Day 20 and samples from 6 randomly selected exposure vessels from the treatment group were analyzed for atrazine concentrat ion.

At the end of exposure period on Day 21, frogs were again weighted and measured. Frogs were subdivide into three groups (n=20

- 22 frogs per group). One group was euthanized and necropsied, one
was selected as the Bd exposure group and one served as the post atrazine exposure Bd negative27 control group. Bd exposure occurred on Day 22 and every eighth day of the next 25

– day period (4 Bd exposures total). Exposure to Bd was through cultured Bd distributed in 50 uL aliqu ots into each control vessel. Concentration of zoospores in the aliquots was measured for each exposure day. Vessels were held upright for the 24 hour bd exposure day, then test solution was replaced and the jars were returned to a slanted position so frog s could exit the water. Frogs were then observed and held until 94 days post initial atrazine exposure (or 74 days post initial Bd

- exposure). At Day 94, all remaining frogs were weighed, measured, euthanized and necropsied for final effects measures. In ad dition to weight and length, The measured biomarkers of health included hepatosomatic index HSI, densities and mean sizes of hepatic and splenic melanomacrophage aggregates (HMAs and SMAs, respectively), density and mean size of hepatic granulomas (HGAs), the neutrophil to lymphocyte ratio (N/L ratio), and immune
function measures including splenosomatic index ( SSI ) , differential leucocyte counts, and the ratio of leucocytes to erythrocytes (WBC/RBC).

To monitor for Bd infection, toe samples from right hin dfoot were collected from each frog killed at Day 94 and examined histologically for Bd and hyperkeratosis. Toe sample from the left hind foot were used for diagnosis and quantification of Bd infection by real time PCR. Full body swabs were also taken at

s everal time points to monitor for infection. As frogs were housed individually, the frequency with which they shed their skin was also quantified as this can be an indicator of Bd infection.

Results : Atrazine was not detected in samples collected from the frogs’ natal pond and none of the frogs initially tested were positive for Bd infection. The concentration of atrazine in the stock solution was 4.28±0.04 ug/L and the water samples collected from the exposure vessel was 1.70±0.26 ug/L.

At the end of th e 21 day exposure period, no difference in SVL or mass was detected in the atrazine exposure group. At the end of the 94 day period, there was a significant difference in mass of the atrazine (11% decrease) and atrazine

- Bd (8% decrease) exposure group from their respective controls. There was no difference in the SVL between treatment and control groups.

Survival was not impacted by atrazine or Bd exposure. No significance difference was found in any biomarkers analyzed, but blood cell ratios wre almost affected by atrazine exposure and occasionally by
Bd exposure.

None of the samples showed microscopic evidence of Bd infection, either through the presence of zoospores or hyperkeratosis and only one sample tested positiv e for Bd via PCR analysis with a low DNA copy number (1.6 DNA copies). Ecdysis (skin molting) was significantly affected by Bd exposure, with Bd exposed frogs shedding skin 1.03±1.03 during the 4 week period and unexposed frogs shedding an average of 0.19± 0.54 times.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment.

Rationale for Use: The study provides information on sublethal effects of atrazine on growth and biochemical parameters in l eopard frogs

. Information on the interaction of atrazine and Bd infection
rate is of limited use.28 Limitations of the Study: Although intended to test the interaction and effect of atrazine on Bd infection, none of the frogs in the study ultimately became infected with Bd . Although some conclusions are drawn in the article on the lack of impact of prior exposure to atrazine on the susceptibility to Bd infection, this conclusion is hard to confirm as Bd infection was not induced in any of the frogs exposed including controls. This could be due to a resistant population of frogs used in the study, the impact of increased skin shedding on disease resistance in the Bd exposed frogs or other factors.

Atrazine was not measured in the control samples. Atrazine i n stock solution and exposure vessels was measured only on Days 20 and 21, respectively.

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division29 Open literature Review Summary

Chemical Name: Atrazine

CAS No: 1912

- 24

- 9

ECOTOX Reference Number and Citation: E160887 Svartz GV; Herkovits J; Perez

- Coll CS

. 2012 Sublethal Effects of Atrazine on Embryo

- Larval Development
of Rhinella arenarum ( Anura: Bufonidae )

. Ecotoxicology 21(4): 1251

- 1259

.

Purpose of Review: Registration Review

Date of Assessment: 4/6/2015

Brief Summary of Study Findings : This study examined the effects of atrazine exposure on embryo

larval d evelopment of the common South American toad, Rhinella arenarum

,

Methods : Three mating pairs of adult common South American toad, Rhinella arenarum weighing approximately 200

- 250 g were acquired in Lobos (B uenos Aires province, Argentina ). O vulation
of females was induced by means of an intraperitoneal injection of a suspension of one homologous hypophysis in 1 mL of AMPHITOX solution (AS) per female

. Oocytes were fertilized in vitro using fresh sperm suspended in AS. Oocytes were fertilized in vitro using fresh sperm suspended in AS. After jelly coat removal, e mbryos were kept in AS and maintained at 20 ± 2 °C. The AS was replaced entirely every three days and monitored weekly to ensure that the pH was at acceptable levels (7 ± 0.5).

For treatments during embryonic stages (S4

- S25), ten embryos were randomly placed in triplicate in 10

cm diameter glass Petri dishes containing 40 mL of test solution. For treatments during prometamorphosis (S28), ten larvae were placed in triplicate in 20 cm

- diameter glass

. Petri dishes containing 100 mL of test solution. Test solutions were entire ly replaced every 48 h. Test groups
included:

l) Continuous exposure from early blastula (S4) up to complete ope rculum stage (S25); 336 h (14 days) at concentrations between 5 and 30 mg/L. 2) Continuous exposure from early complete operculum stage (S25) u p to late complete operculum stage (S25); 336 h (14 days) at concentrations between 1 and 30 mg/L. 3) Continuous exposure from completed back legs (S28) until completion of metamorphosis; 1080 h (45 days) at concentrations between 1 and 30 mg/L. 4) 24 h pu lse

- exposure with concentrations ranging between 1 and 40 mg/L in blastula (S4), neural plate (S1 3), muscular activity (S 1 8), gill circulation (S20), opercular folds (S23), and complete operculum (S25). After the treatments, the embryos were thoroughly was hed and kept in AS until 168 h post

- exposure.

Toxicity tests were performed using technical

- grade atrazine with a purity of 98 %, purchased from Chem Service (West Chester, PA, USA). Due to precipitation of atrazine at higher test concentrations,
0.2% ace tone was added to the test concentrations. Solvent and blank controls were maintained.30 Results: At the blastula stage, sublethal effects were noted in atrazine concentrations over 5 mg/L. Malformations were observed from 48 hours onward and percent affected increased with dose, up to 67% at 30 mg/L. The main malformations were: delayed development, reduced body size, tail and axial flexures, irregular border, different types of edema, wavy tail, microcephaly, malformed mouth and
adhesive structures, gut miscoilin g, underdeveloped gills, cloacal protrusion, an d cellular dissociation

.

D uring short

- t erm chronic (168 h) exposure , t he NOEC value for sublethal effects was 10 mg/L. The percentage of affected embryos was proportional to atrazine concentration, with a maximal of 50 %
affected at 30 mg/L. From 96 h our onwards, malformations, mainly tail, axi al flexures, different types of edema, malformed mouth and adhesive structures, and cellular dissociation in specific regions such as the stunted tail, were observed. In acute exposure, treated larvae showed neurotoxic effects such as spasmodic contraction s and general weakness at concentrations equal or higher t han 15 mg/L. A
reduction in food intake was a l so observed. Lethality was recorded at concentrations higher than 20 mg/L s tarting from 72 h of exposure onwards.

For the 24 hour pulse exposure, lethality in embryos occurred at 40 mg/L 168 hours post exposure. A range of sublethal teratogenic and neurotoxic effects occurred with LOAECs ranging from 15 to >40 mg/L, with the blastula stage being the most sensitive and showing the widest range of eff ects.

Description of Use in Document: Qualitative. This study is of sufficient quality for inclusion in risk

assessment.

Rationale for Use: The study provides information on the potential for teratogenic and neurotoxic
effects due to atrazine exposure during embryo

- larval development.

Limitations of the Study: Wild caught test animals were used for sources of eggs with unknown exposure history.

Author reported that measured concentrations were within 5% of nominal concentration but no measured concentrations were reported or the frequency of measurement. Some concentrations
exceed the water solubility of atrazine (30 mg/L).

Reviewer: Colleen M. Rossmeisl, DVM; ERB3, Environmental Fate and Effects Division