### US ARMY CENTER FOR E NVIRONMENTAL HEALTH RESEARCH 

# Effects of Prochloraz Exposure on Larval and Juvenile Xenopus laev is

Validation of an Amphibian Tier II Assay for Endocrine Disruption

Karen L. Porter, Ph.D., DABT David M. Kumsher March 2012USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

ii 

## TABLE OF CONTENTS

1. INTRODUCTION

................................ ................................ ............................... 1

2. MATERIALS AND METHODS

................................ ................................ ............ 2

2.1. Animals

................................ ................................ ................................ ................................ ............... 2

2.2. Environmental Conditions

................................ ................................ ................................ ................. 3

2.3. Test Chemical

................................ ................................ ................................ ................................ ..... 4
2.4. Experimental Design

................................ ................................ ................................ .......................... 5
2.5. Larval and Juvenile Necropsies

................................ ................................ ................................ .......... 5
2.6. Sex Genotyping

................................ ................................ ................................ ................................ .. 6

2.7. ELISA Assays

................................ ................................ ................................ ................................ ....... 6
2.8. Histology

................................ ................................ ................................ ................................ ............ 7

2.9. Statistics

................................ ................................ ................................ ................................ ............. 7



## 3. RESULTS

................................ ................................ ................................ .......... 7

3.1. Analytical Chemistry

................................ ................................ ................................ .......................... 7
3.2. General Toxicity

................................ ................................ ................................ ................................ . 8
3.3. Sex Phenotype and Genotype

................................ ................................ ................................ .......... 11
3.4. Nuptial Pads

................................ ................................ ................................ ................................ ..... 12
3.5. Gonad Histology

................................ ................................ ................................ ............................... 13

3.5.1 Testes

................................ ................................ ................................ ................................ ......... 13

3.5.2. Ovaries

................................ ................................ ................................ ................................ ...... 15

3.5.3. Oviducts and Wolffian Ducts

................................ ................................ ................................ ..... 16

3.6. Non

- Gonad Histology

................................ ................................ ................................ ....................... 17
3.6.1.Thyroid

................................ ................................ ................................ ................................ ....... 17

3.6.2. Liver

................................ ................................ ................................ ................................ ........... 18
3.6.3. Kidney

................................ ................................ ................................ ................................ ........ 19
3.7. Vitellogenin and T4

................................ ................................ ................................ .......................... 21



## 4. DISCUSSION

................................ ................................ ................................ .. 22

4.1. General Toxicity

................................ ................................ ................................ ............................... 22
4.2. Thyroid Effects

................................ ................................ ................................ ................................ . 23

4.3. Gonad Effects

................................ ................................ ................................ ................................ ... 24

4.4. Recommendation and Conclusions

................................ ................................ ................................ . 25USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

iii 

## ACKNOWLEDGEMENTS

................................ ................................ ..................... 25

REFERENCES

................................ ................................ ................................ ...... 27
List of Abbreviations and Acronyms

................................ ................................ .. 32
Appendix A

– Data Summary Tables

.................................................................. . ... . ..34

Appendix B

– Histopathology Scoring Tables...................................................

.. .....3 7USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

iv 

## LIST OF FIGURES AND TABLES

Figures

Figure 1. Kaplan Meier survival and percent mortality.

................................ ................................ ....... 9 Figure 2. Body Size..............................................................................................................................................10 Figure 3. Liver somatic index

............................................................................................. ................................ 10 Figure 4. Median time to stage

................................ ................................ ................................ ......... 11 Figure 5. Gel image of PCR products from male and female samples.

................................ ................. 12 Figure 6. Microphotographs of fixed larval and juvenile gonads from control animals.

....................... 12 Figure 7. Sex Distribution.

................................ ................................ ................................ ................ 12 Figure 8. Testis histopathology.

................................ ................................ ................................ ........ 14 Figure 9. Gonad histopathology scoring.

................................ ................................ ........................... 15 Figure 10. Ovary histopathology.

................................ ................................ ................................ ...... 16 Figure 11. Oviduct and Wolffian duct histopathology.

................................ ................................ ....... 17 Figure 12. Thyroid gland histopathology

................................ ................................ ........................... 18 Figure 13. Liver histopathology

................................ ................................ ................................ ......... 19 Figure 14. Kidney histopathology

................................ ................................ ................................ ...... 20 Figure 15. Non

- gonad histopathology scoring.

................................ ................................ .................. 21 Figure 16. VTG and T4 levels.

................................ ................................ ................................ ............ 22



## Tables

Table 2

- 1. Larval and Juvenile Feeding Regimen.

................................ ................................ ................. 3
Table 2

- 2. Water quality values.

................................ ................................ ................................ ......... 4
Table 3

- 1. Measured Prochloraz Tank Concentrations by Week of Exposure

................................ ........ 8
Table 1A. Larval and Juvenile Necropsy Somatic Data.

................................ ................................ ...... 35 Table 2A. Larval and Juvenile Sex Data Summary.

................................ ................................ ............. 35 Table 3A. Plasma vitellogenin and thyroxin levels.

................................ ................................ ............ 36 Table 1B. Larval thyroid histopathology scoring.

................................ ................................ ............... 38 Table 2B. Juvenile Liver and kidney histopathology scoring.

................................ .............................. 38 Table 3B. Male histopathology scoring.

................................ ................................ ............................ 39 Table 4B. Female Histopathology Scoring.

................................ ................................ ......................... 40USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

1 1. INTRODUCTION

Prochloraz is an imidazole fungicide used in agriculture as a seed treatment and in foliar spray, which may reach the aquatic environment through agricultural runoff. Prochloraz is registered for use in Europe, Asia, and South Africa and has been found in agricultural surface waters at levels up to 175 μg/L (Bjergager, Hanson et al. 2011) and at detectable levels in groundwater in Norway (Haarstad and Ludvigsen L.G. 2007) . Prochloraz has a high persistence in soil, a low potential for groundwater

exposure, and is considered very toxic to aquatic organisms, with a proposed no observed effe ct concentration ( NOEC ) of 25 μg/L for fish (European Food Safety Authority

, 2011)

. Additionally, t he EU
has rated prochloraz as a substance very toxic to aquati c organisms t hat may cause long

- term effects in
th e aquatic e nvironment (Food and Agricultural Org anization of the Un ited Nations

, 2009)

.

Prochloraz acts as an inhibitor of cytochrome P450 (CYP)

- dependent biosynthesis of ergosterol, a fungal cell membrane component (Henry and Sisler

, 1984)

. Imida zoles strongly interact with the iron component of CY P enzymes, and this interaction is nonspecific, so it can inhibit a large variety of CYP enzymes, including tho se responsible for steroid hormone biosynthesis (Laignelet, Narbonne et al. 1989)

. Prochloraz is a potent aromatase (CYP19) inhibitor with an IC 50 of 0.3 μM in human placental
microsomes (Andersen, Vinggaard et al. 2002) and also acts as a classical androgen antagonist that blocks the androgen receptor (AR) both in vitro and in vivo , but less potently than flutamide (Vinggaard, Nellemann et al. 2002)

. In intact male rats, prochloraz tr eatment reduced seminal vesicle weight and circulating luteinizing hormone (LH) levels, and in the Hershberger assay, prochloraz also reduced ventral prostate weight and weights of other male accessory sex organs (Vinggaard, Nellemann et al. 2002)

. Additionally, male offspring f rom prochloraz treated dams have exhibited decreased circulating and testicular testosterone levels and increased nipple retention (Vinggaard, Christiansen et al. 2005)

.

The effects of prochloraz exposure on amphibians have not been extensively studied. A Danish study using Xenopus laevis embryos in the FETAX assay yielded a no observed effect concentration (NOEC) value of 2 mg/L for growth inhibition and malformations

, and 3 mg/L for mortality (Larsen J and
Sørense n I ,2004) . A longer term larval study using Rana temporaria showed that prochloraz exposure induced a significant increase in proportion of phenotypic males and a decrease in circulating
testosterone levels (Brande

- Lavridsen, Christensen

- Dalsgaard et al. 2008)

. Prochloraz exposure has been shown to decrease vitellogenin (VTG) levels in fathead minnows (Ankley, Jensen e t al. 2005; Ankley,
Benci c et al. 2009; Jensen and Ankley

, 2006; Skolness, Durhan et al. 2011; Thorpe, Pereira ML et al. 2011) , zebrafish (Kinnberg, Holbech et al. 2007; Thorpe, Pereira ML et al. 2011) , and rainbow trout (Sturm, Cravedi et al. 2001)

.
The present study was conducted to determine the effects of prochloraz exposure on larval and juvenile Xenopus laevis and as part of a n interlaboratory v alidation study for the Tier II amphibian assay for endocrine disruptor exposure. Initiation of X. laevis embryo exposure began prior to organogenesis in order to capture any effects the prochloraz may have had on organogenesis and metamorphosis, andUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

2 conti nued 8 weeks past completion of metamorphosis in order to find any effects on early gonad development.

2. MATERIALS AND METHODS

2.1. Animals

Animals used were the African clawed frog, Xenopus laevis , obtained from NASCO (Fort Atkinson, WI)
and allowed to acclimate in our facility for 30 days before use . Breeders were housed in partially filled 120 L tanks with a flow through rate of 5.4 turnovers per day. The aquaculture water used for housing and exposure of the frogs was prepared by mixing rev erse osmosis treated tap water with ground water at approximately a 50:50 ratio. This dilution was done to reduce the natural hardness of the ground water used for the study. The aquaculture water was then aerated, 10 μM filtered, UV sterilized and
heated to 21 °C before use. Adult frogs were fed Nasco pellets (Fort Atkinson, WI) three times per week

. A 12:12

- hour light:dark cycle was maintained.

Embryos were obtained as previously described with the following modifications (Knechtges, Sprando et al. 2007) . Briefly, five breeding pairs were induced to mate using one injection of 500 IU human Chorionic Gonadotropin (Chorulon, Provet, Kansas City, MO) for males and 750 IU for females, and kept overnight in the dark in 4 L breeding chambers in a 21 ± 2 ° C incubator. The next morning, the most viable spawn was rinsed in 2% L

- cysteine at pH 8 in order to remove the gelatinous coatings. Necrotic eggs and n on

- developing or abnormally

- devel oping embryos were removed

. W hen most embryos were
at Nieuwk oop and Faber (NF) stage s 8

– 9 (Hubrecht

- Laboratorium, Nieuwkoop et al. 1967) the embryos were randomly transferred into small beaker s corresponding to each tank, which were then placed
inside the exposure tanks.

A preliminary feeding study was conducted prior to the prochloraz exposure using X. laevis embryos from stage 8 through 66 to determine the optimal feeding re gimen under our conditions. Several feeding regimens were teste d in parallel and the following regimen had low mortality combined with
the least malformation incidence

. Tadpoles were fed 4.5 mL Sera Micron (Pondside Herp Supply, Indian Harbor Beach, FL) suspended in water (22 g per 1L water) twice per day through day 10 of exposure

, increased to 9 mL from da y 11 through 17, increased to 18 mL from day 18 through 43, decreased to 10mL from day 44 through 51 and discontinued on day 52. Half a gram of crushed Xenopus Express pellets ( Brooksville, FL ) were added once per day on days 36 through 40

, increased to twice per day on days 41 through 51, then 1 g whole pellets were fed twice per day from day 52 through completion of metamorphosis. Post metamorphic froglets were fed 2 g pellets once per day from day 56 through 58, 3 g pellets once per day from day 59 through 61. After the tanks were culled down to 10 frogs per tank, feeding was decreased to 2 g pellets once per day from day 62 to 70, increased to 3 g pellets per day from day 71 through 91, and maintained at 4 g once per day from day 92 through the final necropsy (Table 2

- 1 )

.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

3 Table 2

- 1 . Larval and Juvenile Feeding Regimen. Sera Micron suspension was given twice per day and Xenopus Express pellets were given as indicated.

Days Post Hatch Sera Micron (mL) Xenopus Express Pellets (g)
4

- 10 4.5 11

- 17 9 18

- 35 18 36

- 40 18 0.5 x1 a
41

- 4 3 18 0.5 x2 a
44

- 51 10 0.5 x2 a
52

- 55 1.0 x2 56

- 58 2.0 x1 59

- 61 3.0 x1 62

- 70 b 2.0 x1 71

- 91 3.0 x1 92

- final necropsy 4.0 x1

a Pellets lightly crushed. b Tanks culled of frogs in excess of 10 frogs per tank.

Frogs being culled and moribund frogs were euthanized by immersion for 10 minutes in 0.2% Tricaine methanesulfonate (MS

- 222, Sigma, St. Louis, MO) adjusted to pH 7.0 with sodium bicarbonate (Sigma). Frogs euthanized for the larval and juvenile necropsies were first immersed in MS

- 222 solution until a deep surgical level of anesthesia was reached, about 3 minutes, followed by blood collection and euthanasia by ex sanguination and/or cardiac excision

.

2. 2. Environmental Conditions

The exposure was conducted in a fl ow through diluter system with 9 L glass aquaria containing 7 L aquaculture water set i n a water bath maintained at 21° C. Each diluter pump dispensed 220 mL aquaculture water to four replicate a quaria every five minutes with or without prochloraz at 6.7, 20, 60, and 180 μg/L, to equal a flow rate of 9.0 tank turnovers per day. Diluter pump calibration was checked weekly and recalibrated as needed. All tubing used was polytetrafluoroethylene (PTFE ). In order to
minimize microbial co lonization and degradation of prochloraz , well water was 0.2 μm filtered at the diluter inlet, the diluter system was disinfected with 5% Minncare cold sterilant (Mar Cor, Skippack, PA) prior to exposure initiation , and aquaria were replaced during the exposure after completion of metamorphosis

.
Animals were kept on a 12:12 light:dark cyc le, with lighting provided by 32 watt full spectrum bulbs ( Vita

- Lite Plus

, Rexdale, ON, Canada ) providing an inten sity range between 50 0 to 9 00 lux. Water quality was maintained in accordance with ASTM standard E 1241

- 98 (American Society for Testing and Materials International ,2004) and measured weekly. Water quality readings d uring the study ranged from 20.3 to 21.8°C for temperature, 2.7 to 5.5 mg/L for dissolved oxygen, 741 to 962 Siemens/cm for
cond uctivity, 7. 01 to 7.41 for pH, 124 to 137 mg/L (as CaCO 3 ) for alkalinity, 0.07 to 1.31 mg/L for ammonia, and 19 6 to 218 mg/L (as CaCO 3 ) for hardness (Table 2

- 2 ).USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

4 Table 2

- 2 . Water quality values.

Parameter Set Point Mean Minimum Maximum N Temperature (°C) 21 21.3 20.3 21.8 336 pH 7.5 7.21 7.01 7.41 336 Dissolved Oxygen (mg/L) 3.4

– 8.5 4.1 2.7 a 5.5 336 Alkalinity as CaCO 3 (mg/L) >50 b 128 124 137 28 Hardness as CaCO 3 (mg/L) >175 b 206 196 218 28 Conductivity (Siemens/cm ) 500

– 1000 b 798 741 962 336 Ammonia Nitrogen (mg/L) < 1.8 0.53 0.07 1.31 28

a Tanks drained and refilled with fresh aquaculture water. b (Green

, 2010)

2.3. Test Chemical

Prochloraz (Chem Service, Westchester, PA) has low solubility in water, 34 mg/L (Nielsen, Nielsen et al. 2004) , which necessitated stock preparation in a saturation jar (Kahl, Russom et al. 1999)

. Two stock
solution s containing 8.0 and 0.75 mg/L prochloraz were prepared by dissolving 160mg or 15mg prochloraz in 20 mL HPLC grade acetone (Si gma) and adding this solution to a 20 L gl ass carboy (Corning Life Sciences, Lowell, MA ). The carboy was turned on its side and rotated while the acetone evaporated under nitrogen

. Once the acetone had evaporated, 20L Milli

- QTM (Millipore, Billerica, MA ) pu rified water
and a 2

- inch dumbbell style stir bar was added . The carboy was placed on a heater

- stir plate set to 200 rpm and 60°C, which yielded a constant solution temperature of 30°C. After at least 24

– 48 hours stirring, samples were taken from the sto cks and analyzed for prochloraz concentration. If the solutions were within ±10% of target (8.0 ± 0.8 mg/L or 0.75 ± 0.075 mg/L ) the solutions were put into use in the diluter system. The 8.0 mg/L stock was diluted for the 180μg/L and 60 μg/L concentration s, and the 0.75 mg/L stock was diluted for the 20 μg/L and 6.7 μg/L concentrations. Diluter calibration was adjusted
based on measured tank concentrations to compensate for degradation of prochloraz in the tank water.
Prochloraz stock solutions were kept under constant stirring and heating at all times in order to maintain
consistent concentrations.

Samples from each tank were taken weekly and analyzed for prochloraz concentration. Samples containing expected p rochloraz concentrations from 6.7 to 20 μg/L were concentrate d using a C

- 18 solid phase extraction cartridge (J.T. Baker, Phillipsburg, NJ) and eluted with methanol ( Optima grade, Fisher Scientific, Pittsburg, PA) . The eluate wa s diluted 3 0 :7 0 with deionized water then injected o nto a high performance liquid chromatograph (HPLC) equipped with a diode array detector (Agilent 1100 series, Agilent, Palo Alto, CA) and a Zorbax SB

- C18 column ( Agilent, Palo Alto, CA )

. Mobile phase was 70:30 methanol : water, the flow rate was 0.3 mL pe r minute, the injection volume was 100 μl , and
absorbance was read at 205 nm

. Water samples where prochloraz concentrations were expected to be
from 20 to 200 μg/L were analyzed by direct injection. Samples at concentrati ons higher than 200 μg/L
were diluted 8 0 :2 0 with m ethanol. Six prochloraz standards prepared in 20% methanol from 10 μg/L to

200 μg/L were used to generate a standard curve for determination of prochloraz concentrations by linear regression

. The prochloraz detection limit 13.21 μg/L with a relative standard deviation of 7.16 percent and a 95% confidence limit 13.88 μg/L (upper limit) 12.53 μg/L (lower limit).USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

5 2.4. Experimental Design

The animals were exposed to 6.7, 20, 60, and 180 μg/L prochloraz from NF stage 8 until 10 weeks post median time to stage 62 (1 6 weeks of exposure ) . There were eight replicate u nexposed control tanks and four replicate tanks per prochloraz concentration. Each tank initially contained 30 embryos. Seven stage 62 tadpoles per tank were randomly sub

- sa mpled during weeks 5

– 7 of exposure, each tank was culled down to 10 animals per tank on week 9, and the rema ining animals were sampled at 16 weeks of exposure. Prochloraz concentrations were chosen for reproductive effects based on studies using Rana

tem poraria (Brande

- Lavridsen, Christensen

- Dalsgaard et al. 2008)

, zebrafish (Kinnberg, Holbech et al. 2007) , fathead minnow (Ankley, Jensen et al. 2005) , and through preliminary range find studies performed by our collaborating laboratory (data no t shown)

.

2.5. Larval and Juvenile Necropsies

Sampling for the larval nec ropsy took place during weeks 5

- 7 of exposure . On t he first day that an NF stage 62 froglet was seen (day 38 of exp osure) the number of NF stage 62 froglets was counted in each
tank. A random number from 1 to 6 was generated by throwing a d ie, then that number of stage 62 froglets was randomly taken from each tank for euthanasia and necropsy. Thi s was repeated each day
until seven froglets per tan k h ad been taken, a period of 11 days. The froglets selected for sampling

were immersed in a sodium bicarbonate buffered 0.03 % MS

- 222 solution (pH 7) until a deep surgical level of anesthesia was reached. The froglets were weighed and body length from snout t o vent was measured. Blood was collected in a heparinized capillary tube (Fisher, Pittsburgh, PA) after cardiac nicking and sex phenotype was determined by examination of gonads under a dissection microscope; ovaries were distinguished by the presence of lobes interspersed with melanin, and testes were distinguished by their shorter length and lack of pigment (Hayes, Stuart et al. 2006) . The gut was removed and discarded, the legs were removed and stored at

- 20° C for later genomic DNA extraction,
and the carcass was fixed in Modified Davidson’s Fixative (30% ethanol, 20% formalin, 10% glacial acetic acid, 10% glycerol, 30 %. water ). After 4 days, fixed tissue was rinsed in water, and then stored in 10%
normal buffe red formalin (NBF; Sigma). The blood was centrifuged for 10 minutes at 10,000 g at 4° C and the plasma was separated and stored at

- 80° C until analysis. After completion of the larval sampling, stage 62 and 66 froglets continued to be counted until 95% of t he animals in each tank had completed metamorphosis. At week 8 of exposur e, tadpoles and froglets still below stage 62 were euthanized, and at week 9 of exposure, any tank with more than 10 froglets was randomly culled down to 10 froglets per
tank by eutha nasia of the excess froglets.

Sampling for the juvenile ne cropsy took place over three days during week 16 of exposure

. Since the duration of the necropsy was brief, all of the frogs from each tank were taken at once, but the order of tanks was randomized. Each frog was immersed in a sodium bicarbonate buffered 0.2% MS

- 222 solution (pH 7) until a deep surgical level of ane s thesia was reached. The frog s were weighed and body length
from snout to vent was measured. Blood was collected in heparinized M icrotainers TM (BD Biosciences
, Franklin Lakes, NJ ) by foot amputation and the foot was stored at

- 20° C for later genomic DNA

extr action. S ex phenotype was determined b y gross examination of gonads and t he liver was remov ed and weighed. The forelimbs were examined for the presence of nuptial pads under 10X magnification.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

6 After weighing and removal of the gut and hind legs, the liver and carcass were fixed in Modified
Davidson’s Fixative. After 3 to 5 days, Davidson’s fixed tissue was rinsed in water, and then stored in
10% NBF. The blood was ce ntrifuged for 5 minutes at 10,000 g at 4°C, and the plasma separated and
stored at

- 80° C un til analysis.

2.6. Sex Genotyping

Genomic DNA was extracted from the toe tissue using Qiagen DNeasy blood and tissue kits (Qiagen, Valencia, CA ) according to manufacturer instructions. The isolated DNA was amplified by PCR using a Qiagen Multiplex PCR kit and primers for DM

- W , an ovarian development gene present in all X. laevis females, and DMRT1 , a gene present in both male and female X. laevis

, used as a control. The DM

- W primer sequences were 5’

- CCACACCCAGCTCATGTAAAG

- 3’ and 5’

- GGGCAGAGTCACATATACTG

- 3’ and the

DMRT1 primer sequences were 5’

- AACAGGAGCCCAATTCTGAG

- 3’ and 5’

- AACTGCTTGACCTCTAATGC

- 3’ (Yoshimoto, Okada et al. 2008)

. PCR products were run on a 2% agarose E

- GelTM (Invitrogen, Carlsbad, CA), visualized using a VersadocTM system (Bio

- Rad, Hercules, CA ) , and photographed . The gels were

exa mined for DMRT1 control bands (206 bp) and the DM

- W bands (260 bp) and any sample that did not
have the control band was repeated. The presence of both the control and DM

- W bands indicated a female genotype, and the presence of the control and absence of DM

- W indicated a male genotype.

2.7. ELISA Assays

ELISA assays for vitellogenin were performed as previou sly described (Olmstead, Korte et al. 2009)

. Briefly, 96

- well ELISA microtiter plates were coated with purified X. laevis vitellogenin overnight, and then normal goat serum was used to block nonspecific sites. Primary anti

- vitellogenin serum preabsorbed with normal male X. tropicalis serum was diluted 1:2 ,000 in assay buffer and plasma samples were dilut ed 1:300 in assay buffer . A standard curve was prepared with purified vitellogenin diluted with assay buffer to 800, 400, 200, 100, 50 and 25 ng/mL. Samples an d standards were
i ncubated with primary antibody and added to the plate in duplicate. Goat anti

- rabbit IgG conjugated
wi th horseradish peroxidase (Biorad ) was used a s a secondary antibody at a 1:16 ,000 dilution. Plates were incubated with tetramethylbenzid ine substrate ( TMB; Sigma ) and the reaction was stopped after

2 0 min w ith 1 N phosphoric acid. Absorbance was measured at 450 nm in a plate reader. Samples with values above the highest standard were repeated at a higher dilution. Values below the lowest st andard were reported as calculated if they were within the 95% confidence interval ( CI) of the lowest standard,

as one

- half the lower bound of the lowest standard 95% CI if the value is between the lowest standard’s lower bound and the Bo upper bound, or a s zero if within the 95% CI of specific binding ( Bo )

.
ELISA assays for plasma thyroxin (T4) were performed using a commercial total T4 kit (MP Biomedicals

, Solon, OH ) using the manufacturer instructions with the following modifications. A stock T4 standar d was made by dissolving 25 mg L

- thyroxin e (Sigma) in 25 mL 0.1 N sodium hydroxide and w orking T4
standards were diluted in a solution of 10% charcoal stripped X. laevis plasma in Low Cross Buffer (Candor Bioscience, Wangen, Germany) , to 333, 111, 37, 12.3, and 4.1 ng/mL. All samples were diluted
1:10 in Low Cross Buffer

, and samples and standards were added to the T4 primary antibody coated plate in duplicate. Kit T4

- HRP conjugate was added and the plate was incubated for one hour at roomUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

7 temperature. The plate was washed in deionized water, incubated with TMB substrate for 20 minutes, and the reaction was stopped with 1M hydrochloric acid. Absorbance was measured at 450 nm in a plate reader. Due to the low sample volume from the larval necropsy, these samples were pooled by tank, taking an equal volume from each sample for the pool, prior to analysis. Samples with values above the highest standard were repeated at a higher dilution. Values below the lowest standard were reported as calculated if they we re within the 95% confidence interval (CI) of the lowest standard, as

one

- half the lower bound of the lowest standard 95% CI if the value is between the lowest standard’s lower bound and the Bo upper bound, or as zero if within the 95% CI of specific bindi ng (Bo).

2.8. Histology

All tissue samples were fixed in Modified Davidson’s Fixative for 3

– 5 days, then rinsed in water and
stored in 10% NBF until tissue processing. Larval carcasses were cut transversely posterior to the
forelimbs and anterior to the pelvis for the gonad and cut transversely through the shoulder joint and anterior to the eyes for the thyroid. The thyroids were placed into cassettes and sent to a contract laboratory for pa raffin embedding, sectioning, hemat oxylin and eosin (H&E) stainin g, and histological examination, and the gonads were put into long term storage in 10% NBF

. Juvenile livers were put into cassettes and sent for processing. Juvenile carcasses were cut transversely across the abdomen anterior and posterior to the testis or immature ovary for the gonad, and cut transversely through the shoulder joint and anterior to the eyes for the thyroid. The gonads were placed into cassettes and sent to a contract laboratory for processing, and the thyroids were put into long term storag e in 10% NBF.

2.9. Statistics

Data were c alculated from the means of eight replic ate tanks for controls and four replicate tanks for exposed groups and analyzed using ANOVA with Dunnett’s t

- test, T amhane

- Dunnett, T3 test
, or from
the Mann

- Whitney

- Wilcoxon U test when there was heterogeneity of variance from Levine’s test

. Except for mortality, two tanks from the control group and one tank from the 20 μg/L prochloraz group which had >20% mortality were omitted from statistical analysis. Mortality and time t o metamorphosis data were analyzed as events using Kaplan

- Meier log rank analysis. Data for plasma VTG and T4 levels were log transformed prior to analysis. Data for proportion of females and nuptial pad presence were arcsine transformed prior to analysis. P values ≤ 0.05 were considered significant. Statistics were run using SYSTAT v. 12 (SYSTAT Inc., Chicago IL) and SPSS v. 16 (SPSS Inc., Chicago IL). All data are presented as plus or minus the standard deviation except where noted.

3. RESULTS

3.1. Analyt ical Chemistry

Samples for prochloraz chemical analysis were collected weekly from the tanks and from each stock 24

– 48 hours after preparation

. Averaged across the entire study, tank prochloraz concentration s were 6.6 ± 0.4, 21 ± 2, 60 ± 5, and 176 ± 11 μg/L for the nominal values of 6.7, 20, 60, and 180 μ g/L, respectively (Table 3

- 1 ). Concentration variations ±10% of nominal were considered acceptable, and diluter calibration was adjusted to compensate for concentrations falling outside this range. Tanks wereUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

8 changed out after completion of metamorphosis to remove organic and microbial material which may have contributed to excess degradation of prochloraz in the tanks.

Table 3

- 1 . Measured Prochloraz Tank Concentrations by Week of Exposure Measured Prochloraz Concentration (μg/L) Week 6.7 20 60 180 Action 0 6.4 ± 0.5 17 ± 0.4 62 ± 1 182 ± 3 1 7.1 ± 0.1 21 ± 0.2 63 ± 1 187 ± 2 2 7.2 ± 0.1 22 ± 1.6 65 ± 1 178 ± 3 3 6.9 ± 0.1 22 ± 0.4 64 ± 1 192 ± 4 4 6.7 ± 0.3 22 ± 0.5 66 ± 1 184 ± 2 5 6.6 ± 0.3 17 ± 0.3 57 ± 1 170 ± 2 6 6.1 ± 0.3 16 ± 0.7 61 ± 2 179 ± 1 7 6.8 ± 0.3 21 ± 0.6 64 ± 3 182 ± 3 8 6.3 ± 0.3 21 ± 0.6 61 ± 2 178 ± 3 9 6.3 ± 0.3 21 ± 0.3 61 ± 0 175 ± 2 Replaced all tanks 10 6.4 ± 0.2 20 ± 0.9 55 ± 5 170 ± 2 11 7.3 ± 0.6 23 ± 0.4 60 ± 2 169 ± 3 12 7.0 ± 0.2 25 ± 0.6 56 ± 3 166 ± 4 13 6.1 ± 0.3 22 ± 0.9 54 ± 2 160 ± 5 14 6.3 ± 0.3 21 ± 0.4 53 ± 3 177 ± 4 15 6.0 ± 0.7 19 ± 1.4 55 ± 1 181 ± 17 16 6.4 ± 0.4 19 ± 1.6 50 ± 2 146 ± 3 Mean 6.6 ± 0.4 21 ± 2.3 60 ± 5 176 ± 11 Range 5.3

– 8.2 15

– 25 49

– 68 142

– 206 N 18 18 19 19

3.2. General Toxicity

Percen t spontaneous mortality in all tanks was 17.5%, 16.7%, 21.7%,7.5 %, and 1 0.0% for unexposed controls, 6.7, 20, 60, and 180 μg/L prochloraz exposed animals, respectively. There were two tanks in the control group and one tank in the 20 μg/L group that had >20% mortality. These tanks were excluded from data analysis for the other endpoints. Tot al spontaneous mortality was 15.1 % including
all groups through the end of the study , 81% of spontaneous deaths occurred during the first 10 days of

exposure, and there wer e no spontaneous deaths after 56 days of exposure. A Kaplan

- Meier log rank analysis was performed, treating accidental deaths and eut hanasia deaths for interim and final necropsies as censored data (Fig. 1 A , B ). There was no significant difference between control and prochloraz exposed groups, indicating that there was no signific ant mortality associated with prochloraz

exposure.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

9 Figure 1. Mortality. Kaplan Meier survival including all tanks (A) and percent mortality excluding tanks with >20% mortality (B).

Frogs in the larval and juvenile necropsies were weighed and measured for body le ngth from sn out to vent. Larval lengths and weights for both male and female froglets in prochloraz exposed groups were similar to weight s in une xposed controls (Fig. 2A

, B )

. Juvenile lengths and weights for both male and female frogs were significantly decreased in t he 180 μg/L prochloraz group com pared to the controls (Fig. 2 C, D ), p≤0.05

. Juvenile liver somatic indices (LSI) for both male and female frogs were significantly decreased in the 60 and 180 μg/L prochloraz group s , and significantly decreased in females from the 20 μg/L group, compared to the controls (Fig. 3 )

. Juvenile f rogs in all groups did not display species characteristic sexual dimorphism in body s ize , probably due to immaturity . These data indicate that prochloraz exposure had no effect o n body growth, sexual dimorphism, or size a t stage 62 of metamorphosis, but higher prochloraz concentrations may retard growth and reduce body size later in development

. Days of Exposure 0 20 40 60 80 100 120Survival

0.0 0.2 0.4 0.6 0.8 1.0

60 μ g/L
6.7 μ g/L
20 μ g/L
180 μ g/L Control

Prochloraz μ g/L 0 6.7 20 60 180Percent Mortality

0 5 10 15 20 25 

### A BUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

10 Figure 2. Body Size. Larval body weights (A) a nd length s (B), and juvenile weights (C) and lengths (D) separated by male (  ) and female ( ). * Significant difference from control using ANOVA with Dunnett’s t, p≤0.05.

Figure 3. Liver somatic index (LSI)

. M ale (  ) and female ( ). * Significant difference from control using ANOVA with Dunnett’s t, p≤0.05.Body Weight (g)

0.0 0.2 0.4 0.6 0.8 1.0Body Weight (g)

0 5 10 15 20 25

Prochloraz μ g/L 0 6.7 20 60 180Body Length (mm)

0 5 10 15 20

Prochloraz μ g/L 0 6.7 20 60 180Body Length (mm)

0 20 40 60 

### A

B C

D * *

* *

Prochloraz μ g/L 0 6.7 20 60 180Liver Somatic Index

0 1 2 3 4 5 6 7



### * * * * *USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

11 The median time to stage 62 was 45 days in the control group and this time was used to calculate the week for the juvenile necropsy, i.e. ten weeks post 45 days (Fig 4 ) . Stage 62 froglets were first seen on day 38 and the last seen on day 56

. There was no significant difference between controls and the prochloraz exposed groups for median time to stage 62. The median time to stage 66, completion of metamorphosis, was 50 days in the control group. Stage 66 froglets were first seen on day 4 1 and last seen on day 62 (Fig 4 ), after which, any froglets below stage 62 were euthanized. There was no significant difference between controls and the prochlo raz exposed groups for median time to stage 66. These data indicate that prochloraz exposure had no effect on time to metamorphosis.

Figure 4 . Median time to stage

. Stage 62 (  ) and 66 ( ).

3.3. Sex Phenotype and Genotype

Sex phenotype was determine d for both larval and juvenile necropsies by gross examination of the gonad

. Sex genotype was determine d for both larval and juv enile necropsies by PCR for DM

- W , an ovarian development gene only present in female X. laevis (Yoshimoto, Okada et al. 2008) ( Fig. 5) . At stage 62 of metamorphosis, the larval gonad was not sufficiently developed to make an accurate
determ ination of sex ph enotype (Fig. 6 ), so sex reversal, if present, could not be determined. The juvenile gonad, while immature, was sufficiently developed to see distinct testes or ovaries (Fig. 6 ), enabling the determination that there was no sex reversal present in the juve nile sample. Both larval and juvenile sex genotype showed no significant difference between controls and prochloraz exposed groups for percent female or for male/female index (Fig. 7 A, B ). These data indicate that prochloraz exposure had no effect on sex d ifferentiation or juvenile sex phenotype

. Prochloraz μ g/L 0 6.7 20 60 180Days of Exposure

0 10 20 30 40 50 60USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

12 Figure 5. Gel image of PCR products from male and female samples

.

Figure 6. Microphotographs of fixed larval and juvenile gonads from control animals

. Larval gonads are at 24X magnification and juvenile gonads are at 12X magnification.

Figure 7

. Sex Distribution. Percent female (A) and male/female ratio (B) for larval (  ) and juvenile ( ) samples.

3.4. Nuptial Pads

Nuptial pads , a secondary sex characteristic of male X. laevis

, were present in a few male individuals in
control and al l prochloraz exposed groups, but there was no significant difference from controls. Nuptial pads on females were not present in controls and in all but the 180 μg/L prochloraz (4.2 ± 8.3%) exposed Prochloraz μ g/L 0 6.7 20 60 180Percent Female

0 20 40 60 80 100

Prochloraz μ g/L 0 6.7 20 60 180M/F Index

0.0 0.5 1.0 1.5 2.0 2.5 3.0 

### A B DM

- W

DMRT1

M M F F

Larval Male Larval Female Juvenile Male Juvenile FemaleUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

13 group, which represented only 1 individual. The low numbers of males with nuptial pads may be due to the immature state of the animals at time of necropsy.

3.5. Gonad Histology

3.5.1 Testes

Juvenile testes were numerically graded for gonadal degeneration, spermatogonia number, t ubular eosinophilic droplet s, and as ynchronous development (Fig. 8 and 10A). Thinning and loss of the germinal epithelium in the absence of overt cell degeneration, necrosis or tubular collapse was observed with increasing incidence and severity in males with increasing prochloraz dose expos ure, with the 20, 60, 180 μg/L prochloraz exposed groups significantly different from control. In the control group, only 17% had minimal to mild germinal epithelium thinning/loss, while minimal to moderate degeneration was present in 46, 78, 83, and 92% of 6.7, 20, 60, and 180 μg/L prochloraz exposed frogs, respectively. Small punctate, brightly eosinophilic droplets were often observed in tubular lumens and less commonly
betwe en cells within spermatocysts. The controls were minimally affected, and in th e 20, 60, and 180 μg/L groups the effect was significantly higher in the control; 11, 38, 11, 38, and 83% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Asynchronous development, where cells of markedly variable maturity (spermatocyte s, spermatids and spermatozoa) were contained within a single spermatocyst, was observed rarely in the controls, at a low level in lower concentrations

, and was significantly increased in the 180 μg/L prochloraz exposed group; 6, 17, 11, 8, and 50% of unex posed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. A minimal increase in spermatogonia number was seen in a low percentage of the control and lower dose groups, and a significantly higher percentage of the males from the 180 μg/L group; 11, 23, 22, 21, and 58% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively.

Other findings, apoptotic/necrotic gonadal degeneration, interstitial cell hypertrophy/hyperplasia, interstitial fibrosis, mineralization, and m ononuclear cellular infi ltrate , which did not show significant differences from control, were also numerically scored (Appendix B ). Scattered foci of shrunken, hypereosinophilic and/or pyknotic cells characteristic of minimal cell necrosis or apoptosis were present groups with th e smallest incidence in the controls, 22%, and in higher incidences in prochloraz exposed groups, although not concentration dependent; 71, 33, 50, and 67% of 6.7, 20, 60, and 180 μg/L prochloraz exposed frogs, respectively. Minimal interstitial cell hyper trophy/hyperplasia was observed in all groups with the lowest incidence in the controls, 11%, and higher incidences in prochloraz exposed groups, although not concentration dependent; 52, 33, 33, and 50% of 6.7, 20, 60, and 180 μg/L prochloraz exposed frog s, respectively. Minimal to occasionally mild interstitial fibrosis was present in all groups with increasing incidence in prochloraz treated groups, although not concentration dependent; 31, 60, 78, 63, and 50% of unexposed, 6.7, 20, 60, and 180 μg/L expo sed frogs, respectively. Small mineralized foci of minimal severity were present in 11, 23, 44, 38, and 33% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Minimal, or rarely, mild mononuclear cell infiltrates composed of lymphocytes, histiocytes and few plasma cells were present in the testes of animals from all study groups with no concentration dependent relationship; 17, 67, 22, 50, and 17% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

14 Figure 8. Testis histopathology. Control testis at 20X (A) . A seminiferous tubule is outlined in green, individual spermatocysts in black, and small aggregates of interstitial cells in red. Small numbers of spermatogonia (yellow arrows), developing spermatozoa (black arrows), and punctate, brightly eosinophilic droplets or residual bodies (green arrows) are present. Testis from 180 μg/L prochloraz exposed animal at 20X (B) showing gonadal degeneration and eosinophilic dro plet formation. There is thinning / loss of the germinal lining epithelium with frequent exposure of the basal lamina (red arrows) and frequent accumulations of eosinophilic material (green arrows). Testis from 60 μg/L prochloraz exposed animal at 20X (C) showing increased spermatogonia (red arrows) forming small groups and chains within the seminiferous tubules. Testis from 180 μg/L prochloraz exposed animal at 20X (D) showing asynchronous development in a single spermatocyst containing early spermatocyte s (green arrow), late spermatocytes (yellow arrow), and developing spermatozoa (red arrow).



# A B

C DUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

15 Figure 9. Gonad histopathology scoring. Percent juvenile testes (A) with thinning and loss of the germinal epithelium ( ), tubular eosinophilic droplets ( ), asynchronous development ( ), increased spermatogonia ( ), and Grade 2 oviducts ( ). Percent juvenile ovaries (B) with atresia ( )
and interstitial fibrosis ( ). *Significant difference from control , p ≤ 0.05

.

3.5.2. Ovaries

Juvenile ovaries were numerically graded for gonadal degeneration (atresia) , interstitial fibrosis,

m ononuclear cellular infiltrate , and hypoplasia (Fig. 9B and 10) . Almost all examined ovaries were consistent with stage VII ovary differentiation (Ogielska and Kotusz

, 2004) with the majority of the ovary composed of individualized diplotene oocytes separated by a thin band of flattened follicular cells. Minimal to mild ovarian degeneration w ith atresia (apoptosis/necrosis) of individual oocytes was present at 22, 38, 11, 63, and 67% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. In many animals, the atretic follicles were associated with minimal fibrosis or mononuclear or less commonly granulomatous inflammation. Minimal or, rarely, mild or moderate interstitial fibrosis was seen at 32, 46, 22, 75, and 75% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Minimal, or rarely mild, mononuclear cell infil trates composed of lymphocytes, histiocytes and few plasma cells were present frequently in the ovaries of all groups with no concentration dependent relationship; 60, 79, 44, 83, and 67% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Three hypoplastic ovaries were observed in this study, two control animals with ovaries at stage IV and one 60 μg/L prochloraz exposed animal with ovaries at stage V of ovary differentiation. There were no significant differences from control seen in any of the ovarian endpoints. Prochloraz μ g/L 0 6.7 20 60 180Percent

0 20 40 60 80 100 120 140



## *

* * *

* * * *

Prochloraz μ g/L 0 6.7 20 60 180 

## A BUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

16 Figure 10. Ovary histopathology. Control ovary at 20X (A) showing diplotene oocytes separated by a single layer of flattened perifoll icular cells (green arrow) and smaller oogonia and secondary oocytes located along the margin (red arrows). Ovary from 20 μg/L prochloraz exposed animal at 40X (B) showing a collapsed, atretic follicle surrounded and focally infiltrated (red arrow) by mononuclear inflammatory cells. Ovary from 180 μg/ L prochloraz exposed animal at 40X (C) showing ovarian fibrosis. Diplotene follicles ( D ) are separated by several bands of fibrous tissue (red arrows) and the flattened ovarian cavity (black arrows).

3.5.3. Oviducts and Wolffian Ducts

In all females, the oviduct was a distinct tubule lined by cuboidal epithelium with cell debris or mucin filling the lumen, Grade 2 (Fig. 11) . In 44% of control males the oviducts were present only as a discrete fibrous tag with proliferative fibroblasts, Grade 1 (Fig. 11) , and consistent with oviduct regression in male frogs at this stage of maturation (Kelly, 1996). In the majority of control males, 56%, oviduct regression had not taken place and Grade 2 oviduct formation was present, and the percentage of Grade 2 oviducts in the males generally increased with increasing dose exposure; 71, 78, 88, and 92% of 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Wolffian ducts were present in all examined sections, characterized by circular tubules lined by tall columnar epithelium surrounding a large central



# A B

CUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

17 lumen filled with scant basophilic mucin. There were no significant differences from control seen for
oviducts or Wolffian ducts.

Figure 11. Oviduct and Wolffian duct histopatholo gy. Grade 1 oviduct from control male at 40X (A)

. Grade 2 oviduct from control female at 40X (B) . Normal Wolffian duct at 40X (C).

3.6. Non

- Gonad Histology

3.6.1. Thyroid

Larval thyroids were numerically graded for thyroid gland and cellular hypertrophy and atrophy, and
increased follicular lumen. Follicular cell height, shape, p yknosis , k aryomegaly , and colloid quality were quantitatively described (Fig. 12 and 15A)

. Only tissues from the unexposed control, 6.7, and 180 μg/L groups were examined. Thyroids from larval froglets exposed to 180 μg/L prochloraz had significantly increased incidence of mild follicular cell hypertrophy (48%) and hyperplasia (48%) compared to contr ols

at 7 .1 and 3.6 % , respectively. In 180 μg/L prochloraz exposed frogs, mild follicular cell hypertrophy and hyperplasia was associated with thyroid gland hypertrophy and increa sed follicular lumen area in a few
individuals

. The increased incidence of fo llicular cell hypertrophy and hyperplasia in prochloraz exposed
X. laevis were consistent with previous studies where R. temporaria exposed to 252 μg/L prochloraz 

# A B

CUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

18 through metamorphosis showed a significant increase in follicular cell height and pronounced hyperplasia (Brande

- Lavridsen N, Christensen

- Dalsgaard J et al. 2010)

.

Figure 12

. Thyroid gland histopathology. (A) 4X and 40X (B) from an unexposed control frog. Thyroid glands at 4X (C) showing mild follicular cell hypertrophy and hyperplasia affecting both lobes, and at 40X (D) showing mild follicular cell hyperplasia and hypertrophy with pseudostratification from a 180 μg/L prochloraz exposed frog.

3.6.2. Liver

Juvenile livers were numerically gr aded for hepatocellular atrophy and b iliary epithelial hyperplasia

. A random subsample of tissues from each replicate was examined (Fig. 13 and 15B)

. Minimal to severe hepatocellular at rophy was a common finding in prochloraz exposed frogs o ccurring in 21 , 67, and 100 %
of 20, 60, and 180 μg/L prochloraz exposed frogs , respectively. A trophy was no t observed in control or 6.7 μg/L prochloraz exposed frogs. Minimal atrophy was characterized by a decrease in the size of hepatocytes, predominantly around multiple central veins and m ild atrophy involved central vein hepatocytes, mid

- zonal and portal vein hepatocytes

, but generally spared sub

- capsular hepatic cords. Moderate atrophy was more diffuse and involved sub

- capsular hepatocytes in all locati ons . With severe atrophy, all hepatocytes were small and this change was accompanied by a subjective sub

- gross decrease (50

- 75%) in liver size. Hepatic cords with minimal to moderate atrophy were variably thin due
to the variation in hepatocyte size and associated sinusoids were often collapsed and disorganized. Hepatic cords with severe atrophy were more consistently thin due to the diffuse decrease in



# A B

C DUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

19 hepatocyte size; some areas appeared solid due to sinusoidal collapse. Atrophied hepatocytes frequently had lacy vacuolated or glassy eosinophilic cytoplasm. In 20 μg/L frogs, 8% had minimal atrophy, 8% had mild atrophy, and 4% had moderate atrophy. In 60 μg/L frogs, 25% had minimal atrophy, 29% had mild atrophy, 8% had moderate atrophy, and 4% had severe a trophy. In 180 μg/L frogs, 4% had minimal atrophy, 42% had mild atrophy, 38% had moderate atrophy, and 17% had severe atrophy.

Figure 13 . Liver histopatholgy. (A) 4X and 10X (B) from an unexposed control juvenile frog. Liver at 4X (C) and 10X (D) from a 180 μg/L prochloraz exposed frog showing severe atrophy, consistently small hepatocytes, thin hepatic cords, and collapsed sinusoids.

3.6.3. Kidney

Juvenile kidneys were numerically graded for accumulation of glomerular and intratubular protein, renal tubule vacuolization, interstitial fibrosis, and presence of i ntracytoplasmic amphophilic inclusions (Fig. 14 and 15C) . Minimal to mild accumulations of protein within the Bowman’s capsule of the glomerulus
were observed in all groups with a non

- significant trend toward higher percentages in the higher concentration exposed groups; 17, 17, 17, 25, and 38% of unexposed, 6.7, 20, 60, and 180 μg/L expose d frogs, respectively . Minimal intratubular accumulations of protein in sca ttered renal tubules were a consistent finding across all groups without concentration dependent correlation; 31, 33, 22, 25, and 

# A B

C DUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

20 38% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Minimal vacuolization of renal
tubules cells was obse rved in 11% of control animals. Minimal and occasional mild to moderate epithelial vacuolization was seen more frequently in animals exposed to prochloraz (29, 22, 13, and 21% of 6.7, 20, 60, and 180 μg/L prochloraz exposed frogs, respectively), although t here was no concentration dependent correlation or significant difference from control seen. Minimal to mild interstitial fibrosis was present in large numbers of animals in all groups; 45, 67, 50, 54, and 67% of unexposed, 6.7, 20, 60, and 180 μg/L expose d frogs, respectively. A minority of animals had mild fibrosis; 9, 8, 8, 16, and 16% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. While there was a trend toward increased fibrosis in the higher concentration groups, it was not sign ificantly different from controls. Large numbers of renal tubules contained discrete, amphophilic intracytoplasmic bodies (inclusions) in 54% of the 180 μg/L group animals, and in one frog from the 60 μg/L group. These inclusions appeared to be similar to Mallory bodies seen in alcoholic liver disease (Kumar, Abbas et al. 2005)

.

Figure 14 . Kidney histopathology. A n unexposed control frog at 4X (A) showing focal mineralization (green arrow) and small numbers of erythropoietic cells (red arrows) adjacent to a glomerulus. Kidney at 20X from a 180 μg/L prochloraz exposed frog (B) showing a normal renal tubule (T) and tubules with vacuolization (V). Kidney at 20X from a 180 μg/L prochloraz exposed frog (C) showing renal interstiti al fibrosis

. Kidney at 40X from a 180 μg/L prochloraz exposed frog (D) showing intracytoplasmic amphophilic inclusions (red arrows). 

# A B

C DUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

21 Figure 15

. Non

- gonad histo patho logy scoring. Percent larval froglet thyroids (A) with thyroid gland hypertrophy ( ), follicular cell hypertrophy ( ), follicular cell hyperplasia ( ), and increased follicle lumen area ( ). Thyroids from froglets exposed to20 and 60 μg/L prochloraz wer e not scored (ns )

. Percent juvenile frog livers (B) with minimal ( ), mild ( ), moderate ( ), and severe ( ) hepatocellular atrophy. Percent juvenile frog kidneys (C) with accumulations of glomerular protein ( ), and intratubular prote in ( ), renal tubular vacuolization ( ), mild renal interstitial fibrosis ( ), and amphophilic intracytoplasmic bodies ( ). * Significant difference from control using ANOVA with Dunnett’s t, p≤0.05.

3.7. Vitellogenin and T4

ELISA assays for vitellogenin levels were performed on plasma collected from the larval and juvenile necropsies. The larval sample showed no significant difference between controls and prochloraz exposed groups for p lasma vitellogenin levels (Fig 16 A ). In the juvenile sample, plasma VTG for females in the 180 μg/L prochloraz group was significantly increased compar ed to control f emales (Fig. 16 C ), p≤0.05

. Due to the immaturity of the juvenile frogs, the VTG levels were very low compared to VTG levels in previously repor ted in mature animals (Matsumura, Ishibashi et al. 2005; Urbatzka, Bottero et al. 2007) and the species characteristic sex differences in VTG levels were not evident

. These data
indicate that higher concentration prochloraz exposure may affect VTG levels in juvenile females, but not in larval froglets of either sex. 

### Prochloraz μ g/L 0 6.7 20 60 180Percent

0 20 40 60 80



### Prochloraz μ g/L 0 6.7 20 60 180 

### A B

Prochloraz μ g/L 0 6.7 20 60 180Percent

0 20 40 60 80 100 

### C ns ns 

## * *

*USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

22 ELISA assays for T4 levels were performed on plasma collected from the larval and juvenile necropsies. The larval sample , which was pooled by tank, showed no significant difference between controls and prochloraz exposed gro ups for plasma T4 levels (Fig 16B ). The juvenile sample showed no signifi cant difference between controls and prochloraz exposed groups for plasma T4 levels (Fig 16D ). These data indicate that prochloraz exposure has no effect on circulating T4 levels in either larval or juvenile frogs.

F igure 16

. VTG and T4 levels. Plasma VTG in larval (A) and juvenile (C) males (  ) and females ( ). Plasma T4 in larval (B) and juvenile (D) male (  ) and female ( ) samples. Larval samples were pooled by tank for the T4 assay. Data is represented as geometric mean ± SEM. * Significant difference from control using ANOVA with Dunnett’s t, p≤0.05.

4. DISCUSSION

4.1. General Toxicity

In this study, there was no increase in mortality related to exposure to prochloraz. However, there was significant growth retardation, hepatic atrophy, and a trend toward increased number and severity of renal lesions in the higher prochloraz exposed juvenile frogs

. T here was no difference in body size between the control and prochloraz exposed groups i n the larval sampling. In a study using Rana temporaria exposed to prochloraz, an increase in body weight was seen at metamorphosis, and it was attributed to the significant delay in time to metamorphosis seen in prochloraz exposed groups (Brande

Plasma T4

Prochloraz μ g/L 0 6.7 20 60 180T4 (ng/ml)

0 50 100 150 200 250

Prochloraz μ g/L 0 6.7 20 60 180VTG (mg/ml)

0.000 0.001 0.002 0.003 0.004

Prochloraz μ g/L 0 6.7 20 60 180T4 (ng/ml)

0 10 20 30 40VTG mg/ml

0.000 0.005 0.010 0.015 0.020 

### A

B C

D *USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

23 Lavridsen N, Christensen

- Dalsgaard J et al. 2010) . Since there was no delay in time to metamorphosis observed in this study, similarity in body size between control and prochloraz exposed groups may be expected. Additionally, there is evidence that growth in tadpoles and in juveniles is controlled by different hormones, prolactin in tadpoles and growth hormone (GH) in juveniles (Huang and Brown

, 2000a; Huang and Brown

, 2000b; Snyder and Frye

, 1972) , which prochloraz exposure may affect in different ways. In other species, fathead minnows and zebrafish exposed to high concentration prochloraz had decreased body length in juvenile and adult fish (Thorpe, Pereira ML et al. 2011)

. Additionally, s everal rat studies have shown significantly decreased body weight or rate of growth in
castrated male rats (Blystone, Furr et al. 2007) , dams exposed to prochloraz during pregnancy (Blystone, Lambright et al. 2007) and offspring exposed to prochloraz in uter o (Noriega, Ostby et al. 2005)

.
Atrophy and biliary epithelial hyperplasia were the primary histopathological findings in the livers of juvenile frogs exposed to high concentratio n prochloraz. While prochloraz generally inhibit s P450 enzyme biosynthesis , it has been shown to induce CYP1A1, an enzyme implicated in bioactivation of procarcinogens (Shimada, Inoue et al. 2002; Shou, Korzekwa et al. 1994; Yang, Huang et al. 2005) , and hepatic oxidative st ress biomarkers in stickleback (Sanchez, Piccini et al. 2008)

. Additionally,
k etoconazol, a similar azole antifungal drug, has been shown to induce hepatic injury in rats associated with oxidative damage and DNA fragmentation (Amin and Hamza

, 2005)

. The most striking renal lesion associated with prochloraz exposure in this study was the presence of intracytoplas micamphophilic inclusions within the renal tubular epithelium of high dose juvenile frogs which are reminiscent of Mallory bodies seen in alcoholic li ver disease (Kumar, Abbas et al. 2005) . This finding was suggestive of subcellular/cytoskeletal injury to the tubular epithelial cells, consistent with the central role of the kidneys in excretion and storage of prochloraz metabolites in rats (Laignelet, Riviere et al. 1992;
Needham and Challis

, 1991)

.

4.2. Thyroid Effects

Prochloraz has been thought to affect the thyroid indirectly, through p erturbation of the hypothalamic

pituitary

- gonad (HPG) axis and crosstalk between the HPG axis and hypothalamic

- pituitary

- thyroid (HPT )

axes (Ankley, Bencic et al. 2009; Liu, Zhang et al. 2011)

. The complexity of these interactions may yield paradoxical effects such as when prochloraz exposure results in increased whole body T3 in R. remporaria (Brande

- Lavridsen N, Christensen

- Dalsgaard J et al . 2010) and decreased plasma T4 levels in
adult zebrafish (Liu, Zhang et al. 2011)

. However, there were no prochloraz exposure related changes in plasma T4 levels in the current study

. P lasma T4 levels in control stage 62 froglets averaged 29.6 ng/mL, compared to levels averaging 56 ng/mg tissue in stage 61 X. laevis tadpoles (Simon, Tietge et al. 2002)

, averaging 1.1 n g/ m L in Rana catesbeiana froglets

, (Regard, Taurog et al. 1978)

, averaging 4.8 ng/mL in
Rana clamitans froglets (Weil , 1986)

, averaging 2.8 ng/mL in Rana catesbeiana tadpoles (Miyauchi, LaRochelle, Jr. et al. 1977) , and whole body T4 in X. laevis froglets ranging from 0.25

– 1.25 ng/mL (Gancedo, Alonso

- Gomez et al. 1997)

.

T4 levels in adult Bufo marinus have measured as below limits of detection (Regard, Taurog et al. 1978)
and in adult R. catesbeiana at <0.5 ng/mL (Miya uchi, LaRochelle, Jr. et al. 1977) , compared to 124 ng/mLUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

24 for juvenile X. laevis in the current study. The ELISA assay used in this study had a limit of detection of
1.36

– 4.1 ng/mL, and high background from the frog plasma matrix was diminished by dil ution but not
eliminated. For this reason, it is possible that plasma T4 levels determined in this study may be
artifi cially high and not accurately reflect the thyroid hormone status of the animals. The use of solvent extraction and radioimmunoassay used in most of the studies cited above or liquid chromatography

– inductively coupled plasma

– mass spectrometry (LC

– ICP

– MS) may have eliminated the background and increased sensitivity.

4.3. Gonad Effects

The majority of gonad effects in juvenile X. laevis exposed to prochloraz were observed in the testes

, and prochloraz exposure was possibly also associated with interference of oviduct regression. Prochloraz exposure related lesions in the testes encompassed a variety of time

- dependent, non

specific, gener al degenerative lesions: acute (cellular necrosis/apoptosis), sub

- acute (interstitial cell hyperplasia, eosinophilic droplet formation and increased spermatogonia) and chronic (germinal epithelial thinning/loss and fibrosis). While the majority of these ch anges with the notable exception of the germinal epithelial thinning/loss tended to be minimal to mild, the increasing concurrence of these changes with increased prochloraz exposure resulted in testes in the high concentration group that were markedly dif ferent in histologic appearance from those in the control group and which had some combination of at least of three, and frequently more, of these lesions. In those cases with moderate germinal epithelial thinning/loss, the increase in spermatogonia may re flect a relative increase due the
loss of spermatocytes/spermatids rather than an absolute increase in numbers.

The degenerati ve testicular changes in juvenile X. laevis in this study we re consistent with the
anti

- androgenic effects of prochloraz reported in species as divergent as fathead minnows (Ankley, Jensen et al. 2005) and rat (Blystone, Furr et al. 2007) ; specifically, germinal epithelium thinning/loss with high doses have been described with prochloraz exposed rats (Noriega, Ostby et al. 2005) . The
trend toward retention of stage 2 oviducts with increasing prochloraz exposure indicates interference
with normal oviduct re gression in males (Kelly, 1996) which is similar to the feminization of the secondary sexual characteristics of male rats during perinatal proc hloraz exposure (Vinggaard, Christiansen et al. 2005)

.

In females, the only potential prochloraz exposure related lesions were a trend toward increased oocyte atresia (gonadal degeneration) and an increase in interstitial fib rosis in the high concentration exposure

groups. Increases in oocyte atresia have been seen also in prochloraz exposed fathead minnows (Ankley, Jensen et al. 2005) , and zebrafish exposed to prochloraz through development show decreased
number of female s and delay in ovarian development (Kinnberg, Holbech et al. 2007) . The minimal
effect on the ovary compared to the testi s may be related to the additive effects of androgen antagonism and suppression of steroidogenesis on the more androgen sensiti ve testes.

In this study, VTG, an estrogen inducible yolk protein, was significantly in creased in 180 μg/L prochloraz exposed juvenile females , which was unexpected

. In other studies, adult rainbow trout exposed to 100USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

25 μg/L prochloraz had no decrease in V TG (Le, Thomas et al. 2001)

, adult male zebrafish exposed to 16
and 64 μg/L prochloraz show ed a significant in crease in VTG , bu t a significant decrease when exposed to 202 μg/L prochloraz (Kinnberg, Holbech et al. 2007) , and male medaka exposed to 20, 100, and 300 μg/L prochloraz showed a significant increase in VTG, but not consistently (OEC D
, 2006)

. However, adult fathead minnows exposed to 2 0

- 300 μg/L prochloraz showed a significant decrease in VTG (Jensen and Ankley

, 2006)

, female R. temporaria froglets exposed to 252 μg/L prochloraz showed decreased VTG
below limit of detection (Brande

- Lavridsen , Christensen

- Dalsgaard et al. 2008)

. This indicates that VTG response to prochloraz exposure is not uniform across species, sex, and stage of development. The animals exposed in the current study were not sexually mature, which may have been a factor in the unexpected increase in VTG levels seen in female juveniles.

4.4. Recommendation a nd Conclusions

Besides specifically determining the effects of prochloraz exposure on X. laevis , the general intent
behin d this study was to validate a tier II amphibian assay for endocrine disruptors. Thus, practical
considerations of time and resources f igured into the study design. Additional endpoints, such as plasma T3, TSH, estradiol and testosterone levels, and gonad weights may have been useful for evaluating the effects of prochloraz exposure. The timing of the larval sampling at stage 62 was link ed to peak TSH levels, but determination of TSH levels was impractical due to a need for custom antibodies to
X. laevis TSH (Korte, Sternberg et al. 2011) . Timing the larval sampling a t stage 66 would have enabled reliable determination of sex phenotype and collection of larger plasma samples sizes. Matrix effects in
X. laevis plasma hampered ability to reliably determine T4 levels in laboratories without access to radioisotope s or LC

– I CP

– MS (Simon, Tietge et al. 2002)

. Additionally, t he selection of X. la evis as the test species made it impractical to sample at sexual maturity

. However, the study design did enable the detection of thyroid, gonad and general toxicology effects of prochloraz exposure. In the current study,
X. laevis stage 62 froglets expose d to high concentration prochloraz exhibit ed mild thyroid pathology and juveniles exhibit ed mild ovarian pathology, mild to moderate testicular pathology, and in creased
VTG levels consistent with other studies. Since there was evidence of systemic toxicity in high concentration exposed juveniles, interpretation of these results may be complicated

.

ACKNOWLEDGEMENTS

We thank Alan Rosencrance and Bill Dennis for analytical chemistry support , Ron Miller for animal husbandry support (USACEHR) , Erin Lehmer for as sistance with statistic al analysis (Fort Lewis College,
CO ), Sigmund Degitz, Alan Olmstead , Jon Haselman, Lindsey Blake, Joe Korte, and Pat Kosian for
technical direction and advice ( US EPA NHEERL) . This work was supported by US EPA IAG DW

- 21

- 92262901

- 4

. The views, opinions, and/or findings contained in this report are those of the author(s) and should not be construed as official Department of the Army position, policy, or decision, unless so designated by
other official documentation. Citations of commer cial organizations or trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. Research was conducted in compliance with the Animal Welfare Act, and otherUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

26 Fe deral statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (NRC 2011) in facilities that are fully accredited by the Association for Assessme nt and Accreditation of Laboratory Animal Care,
International.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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32 List of Abbreviations and Acronyms



### AR androgen receptor



### °C degree Celsius

CaCO 3 calcium carbonate

CI confidence interval



### CYP cytochrome P450

ELISA enzyme linked immunosorbant assay

F female

FETAX frog embryo Teratogenesis Assay

- Xenopus



### g gram

GH growth hormone

H&E hematoxylin and eosin

HPLC high performance liquid chromatography

HRP horseradish peroxidase

IAG Interagency Agreement

LC

- ICP

- MS liquid chromatography

– inductively coupled plasma

– mass spectrometry



### LH luteinizing hormone

LSI liver somatic index

M male

μg/L microgram per liter

mg milligram

mL milliliter

MS

- 222 t ricaine methanesulfonate

NBF no rmal buffered formalin



### ng nanogramUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

33 

### NHEERL National Health and Environmental Effects Research Laboratory

NOEC no observed effect concentration



### ns not scored

PTFE polytetrafluoroethylene

T4 thyroxine

TMB Tetramethylbenzidine



### TSH thyroid stimulating ho rmone

USACEHR U.S. Army Center for Environmental Health Research

US EPA U.S. Environmental Protection Agency

VTG vitellogeninUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

34 

# Appendix A

Data Summary TablesUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

35 Table 1. Larval and Juvenile Necropsy Somatic Data. Body length is defined as the distance from the tip of the snout to the cloacal vent. Liver somatic index was calculated as organ percent of total body

mass.

Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Larval Sub

- Sample Male Body Mass (g) 0.76 ± 0.0 6 0.72 ± 0.17 0.59 ± 0.1 2 0.71 ± 0.05 0.59 ± 0.13 Male Body Length (mm) 18.5 ± 0.5 18.1 ± 0.9 17.2 ± 0. 1 18.1 ± 0.3 17.1 ± 0.8 * Female Body Mass (g) 0.80 ± 0.1 0.77 ± 0.08 0.66 ± 0. 09 0.75 ± 0.06 0.73 ± 0.07 Female Body Length (mm) 18.9 ± 0. 5 18.9 ± 0.5 18.0 ± 0.6 18.4 ± 0.2 18.3 ± 0.4 Juvenile Sample Male Body Mass (g) 18.2 ± 1. 3 19.4 ± 0.7 18.2 ± 0.8 16.3 ± 0.9 12.3 ± 4.8* Male Body Length (mm) 52.9 ± 0.9 54.0 ± 0.7 5 2.7 ± 0.5 50.9 ± 0.6 45.6 ± 6.3* Female Body Mass (g) 20.0 ± 2. 8 18.0 ± 1.8 20.9 ± 1.3 17.5 ± 2.2 14.1 ± 2.6* Female Body Length (mm) 54.8 ± 2. 9 51.2 ± 3.7 55.8 ± 1.1 52.2 ± 2.5 48.3 ± 4.0*

Organ Somatic Indices Male Liver Index 5.9 ± 0.2 5.3 ± 0.5* 5.4 ± 0. 4 4.9 ± 0.3* 4.1 ± 0.4* Female Liver Index 5.9 ± 0.3 5.7 ± 0.4 5.4 ± 0.1 5.0 ± 0.2* 4.4 ± 0.4*

* Significant difference from control by ANOVA with Dunnett’s t , p ≤ 0.05

.

Table 2

. Larval and Juvenile Sex Data Summary.

Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Larval Subsample

Percent Female 51.8 ± 18 46.4 ± 7 71.4 ± 14 54.9 ± 25 67.9 ± 14 Sex Ratio 1.20 ± 0.98 1.19 ± 0.29 0.44 ± 0.2 9 1.15 ± 1.0 0.52 ± 0.29

Juvenile Sample

Percent Female 51.7 ± 17 40.0 ± 0.0 36.7 ± 6 45.0 ± 13 55.0 ± 17 M/F Ratio 1.1 ± 0.7 1.5 ± 0.0 1.8 ± 0.48 1.4 ± 0.7 1.0 ± 0.9 Male Percent Nuptial Pad s 2.8 ± 6.8 12.5 ± 16 15.1 ± 14 7.1 ± 14 12.5 ± 25 Female Percent Nuptial Pad s 0.0 0.0 0.0 0.0 4.2 ± 8.3USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

36 Table 3 . Plasma vitellogenin and thyroxin levels.

Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Larval Subsample Vitellogenin (μg/ml) Male 2.0 ± 0.5 2.2 ± 0.3 2.4 ± 1.2 2.0 ± 0.4 1.2 ± 0.2 Female 1.8 ± 0.3 2.6 ± 0.9 2.2 ± 0.2 2.7 ± 0.3 2.0 ± 0.6 Thyroxin (ng/ml) All a 29.6 ± 23 16.2 ± 12 56.1 ± 55 14.1 ± 8 39.5 ± 51

Juvenile Sample Vitellogenin (μg/ml) Male 6.1 ± 1.2 8.1 ± 0.9 8.3 ± 2.1 5.4 ± 1.1 17.5 ± 3.6 Female 5.5 ± 1.2 9.1 ± 2.1 6.1 ± 1.3 10.9 ± 2.9 10.7 ± 1.3* Thyroxin (ng/ml) Male 123 ± 54 124 ± 121 102 ± 111 105 ± 98 80 ± 84 Female 124 ± 52 82 ± 98 63 ± 91 86 ± 76 67 ± 56

a Plasma pooled by tank.

* Significant difference from control by ANOVA with Dunnett’s t , p ≤ 0.05

.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

37 

# Appendix B

Histopathology Scoring TablesUSACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

38 Table 1. Larval thyroid histopathology scoring. Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Thyroid Gland Hypertrophy Percent Mi ld 0.0 ± 0.0 0.0 ± 0.0 ns ns 7.1 ± 8.2* Follicular Cell Hypertrophy Percent Mi ld 7.1 ± 14 14.3 ± 11 ns ns 48 ± 18 * Follicular Cell Hyperplasia Percent Mi ld 3.6 ± 7.1 14.3 ± 11 ns ns 48 ± 26 Increased Follicular Lumen Percent Mi ld 0.0 ± 0.0 0.0 ± 0.0 ns ns 7.1 ± 8.2*

n s = not scored

* Significant difference from control by ANOVA with Dunnett’s t , p ≤ 0.05

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Table 2. Juvenile Liver and kidney histopathology scoring. Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Liver Hepatocellular Atrophy Percent All Grades 0.0 ± 0.0 0.0 ± 0.0 21 ± 8.3 63 ± 16 100 ± 0.0

Percent Minimal 0.0 ± 0.0 0.0 ± 0.0 8.3 ± 9.6 25 ± 9.6 4.2 ± 8.3

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 8.3 ± 9.6 29 ± 21 42 ± 32 Percent Moderate 0.0 ± 0.0 0.0 ± 0.0 4.2 ± 8.3 8.3 ± 17 38 ± 25

Percent Severe 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4.2 ± 8.3 17 ± 14 Biliary Epithelial Hyperplasia Percent Minimal 0.0 ± 0.0 0.0 ± 0.0 4.2 ± 8.4 0.0 ± 0.0 4.2 ± 8.4

Kidney Glomerular Protein Percent All Grades 17 ± 15 17 ± 24 17 ± 17 25 ± 17 38 ± 35

Percent Minimal 17 ± 15 13 ± 16 17 ± 17 25 ± 17 38 ± 35

Percent Mild 0.0 ± 0.0 4.2 ± 8.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Tubule Protein Percent Minimal 31 ± 25 33 ± 14 22 ± 19 25 ± 17 38 ± 25 Renal Tubule Vacuolation Percent All Grades 11 ± 8.6 29 ± 16 22 ± 10 13 ± 25 21 ± 16

Percent Minimal 11 ± 8.6 21 ± 16 17 ± 0.0 13 ± 25 13 ± 16

Percent Mild 0.0 ± 0.0 4.2 ± 8.4 5.6 ± 9.6 0.0 ± 0.0 8.3 ± 17 Percent Moderate 0.0 ± 0.0 4.2 ± 8.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Interstitial Fibrosis Percent All Grades 45 ± 17 67 ± 14 50 ± 29 54 ± 8.4 67 ± 24

Percent Minimal 39 ± 14 63 ± 16 39 ± 26 33 ± 19 46 ± 16

Percent Mild 5.6 ± 8.6 4.2 ± 8.4 11 ± 8.4 21 ± 16 21 ± 16 Amphophilic Intracytoplasmic Bodies Percent Minimal 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4.2 ± 8.4 54 ± 37USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

39 Table 3. Male histopathology scoring. Nominal Prochloraz Concentration (μg/L)

0 6.7 20 60 180 Gon adal degeneration (apoptosis/ necrosis ) Percent Minimal 22 ± 26 71 ± 21 33 ± 17 50 ± 58 67 ± 47 Gonadal degeneration (germinal epithelial loss/thinning) Percent All Grades 17 ± 28 46 ± 16 78 ± 50* 83 ± 19* 92 ± 17*

Percent Minimal 17 ± 28 38 ± 8 56 ± 51 58 ± 32 25 ± 32

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 11 ± 19 8.3 ± 17 25 ± 17 Percent Moderate 0.0 ± 0.0 8.3 ± 17 0.0 ± 0.0 17 ± 19 58 ± 17

Percent Severe 0.0 ± 0.0 0.0 ± 0.0 11 ± 19 0.0 ± 0.0 0.0 ± 0.0 Globular intratubular eosinophilic material Percent All Grades 11 ± 16 38 ± 29 11 ± 17 * 38 ± 29 * 83 ± 19 *

Percent Minimal 11 ± 17 15 ± 17 11 ± 19 8.3 ± 19 58 ± 32

Percent Mild 0.0 ± 0.0 23 ± 32 0.0 ± 0.0 29 ± 21 17 ± 19 Percent Moderate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 17 ± 19 Asynchronous development Percent Minimal 5.6 ± 13 17 ± 19 11 ± 17 8.3 ± 17 50 ± 19* Increased spermatogonia Percent Minimal 11 ± 16 23 ± 16 22 ± 19 21 ± 25 58 ± 17* Interstitial cell hyperplasia/hypertrophy Percent Minimal 11 ± 16 52 ± 44 33 ± 32 33 ± 39 50 ± 19 Interstitial fibrosis Percent All Grades 31 ± 19 60 ± 19 78 ± 42 63 ± 29 50 ± 30

Percent Minimal 31 ± 16 60 ± 19 67 ± 33 50 ± 43 50 ± 3 3

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 11 ± 19 13 ± 25 0.0 ± 0.0 Mineralization Percent Minimal 11 ± 25 23 ± 16 44 ± 47 38 ± 8 33 ± 27 Mononuclear cellular infiltrate Percent All Grades 17 ± 18 67 ± 39 22 ± 19 50 ± 33 17 ± 19

Percent Minimal 17 ± 18 60 ± 33 22 ± 19 42 ± 32 8.3 ± 17

Percent Mild 0.0 ± 0.0 6.3 ± 13 0.0 ± 0.0 8.3 ± 17 8.3 ± 17 Oviducts (Type 2) Percent 56 ± 46 71 ± 21 78 ± 19 88 ± 25 92 ± 17

* Significant difference from control by ANOVA with Dunnett’s t , p ≤ 0.05

.USACEHR Tec hnical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

40 Table 4. Female Histopathology Scoring.

Nominal Prochloraz Concentration (μg/L) 0 6.7 20 60 180 Gon adal degeneration (apoptosis/necrosis)

Percent All Grades 22 ± 27 38 ± 29 11 ± 19 63 ± 8.3 67 ± 47

Percent Minimal 20 ± 18 38 ± 29 11 ± 19 63 ± 8.3 67 ± 47 Percent Moderate 5.6 ± 14 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Granulomatous Inflammation Percent Minimal 5.6 ± 14 13 ± 25 0.0 ± 0.0 0.0 ± 0.0 8.3 ± 17 Interstitial fibrosis Percent All Grades 32 ± 21 46 ± 32 22 ± 39 75 ± 32 75 ± 17

Percent Minimal 26 ± 13 46 ± 32 22 ± 39 67 ± 27 75 ± 17

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 8.3 ± 17 0.0 ± 0.0 Percent Moderate 5.6 ± 14 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Mononuclear Cellular I nfiltrate Percent All Grades 60 ± 27 79 ± 25 45 ± 3 9 83 ± 19 67 ± 27

Percent Minimal 49 ± 20 50 ± 43 45 ± 39 71 ± 21 58 ± 21

Percent Mild 11 ± 17 29 ± 21 0.0 ± 0.0 13 ± 25 8.3 ± 17