US ARMY CENTER FOR ENVIRONMENTAL HEALTH RESEARCH

Effects of Prochloraz 

Exposure on Larval and 

Juvenile Xenopus laevis

Validation of an Amphibian Tier II Assay for 

Endocrine Disruption

Karen L. Porter, Ph.D., DABT

David M. Kumsher

March 2012

 

 

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

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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 .................................................................................................25
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ACKNOWLEDGEMENTS .....................................................................................25

REFERENCES......................................................................................................27

List of Abbreviations and Acronyms..................................................................32

Appendix A – Data Summary Tables.........................................................................34

Appendix B – Histopathology Scoring Tables..........................................................37
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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

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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 effect 

concentration (NOEC) of 25 μg/L for fish (European Food Safety Authority, 2011). Additionally, the EU 

has rated prochloraz as a substance very toxic to aquatic organisms that may cause long-term effects in 

the aquatic environment (Food and Agricultural Organization of the United Nations, 2009).

Prochloraz acts as an inhibitor of cytochrome P450 (CYP) -dependent biosynthesis of ergosterol, a fungal 

cell membrane component (Henry and Sisler, 1984). Imidazoles strongly interact with the iron 

component of CYP enzymes, and this interaction is nonspecific, so it can inhibit a large variety of CYP 

enzymes, including those responsible for steroid hormone biosynthesis (Laignelet, Narbonne et al. 

1989). Prochloraz is a potent aromatase (CYP19) inhibitor with an IC50 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 treatment 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 from 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 

Sørensen 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 et al. 2005; Ankley, 

Bencic 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 an interlaboratory validation 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, and 
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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continued 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 reverse 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 non-developing or abnormally-developing embryos were removed. When most embryos were 

at Nieuwkoop and Faber (NF) stages 8 – 9 (Hubrecht-Laboratorium, Nieuwkoop et al. 1967) the embryos

were randomly transferred into small beakers 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 regimen under our conditions. Several

feeding regimens were tested 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 day 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). 
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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 x1a

41-43 18 0.5 x2a

44-51 10 0.5 x2a

52-55 1.0 x2

56-58 2.0 x1

59-61 3.0 x1

62-70b 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 exsanguination and/or cardiac excision.

2.2. Environmental Conditions

The exposure was conducted in a flow through diluter system with 9 L glass aquaria containing 7 L 

aquaculture water set in a water bath maintained at 21°C. Each diluter pump dispensed 220 mL 

aquaculture water to four replicate aquaria 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 colonization 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 cycle, with lighting provided by 32 watt full spectrum bulbs 

(Vita-Lite Plus, Rexdale, ON, Canada) providing an intensity range between 500 to 900 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 during 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 

conductivity, 7.01 to 7.41 for pH, 124 to 137 mg/L (as CaCO3) for alkalinity, 0.07 to 1.31 mg/L for 

ammonia, and 196 to 218 mg/L (as CaCO3) for hardness (Table 2-2).
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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 2.7a 5.5 336

Alkalinity as CaCO3 (mg/L) >50b 128 124 137 28

Hardness as CaCO3 (mg/L) >175b 206 196 218 28

Conductivity (Siemens/cm) 500 – 1000b 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 

solutions containing 8.0 and 0.75 mg/L prochloraz were prepared by dissolving 160mg or 15mg 

prochloraz in 20 mL HPLC grade acetone (Sigma) and adding this solution to a 20 L glass 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) purified 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 stocks 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 concentrations, 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 prochloraz concentrations from 6.7 to 20 μg/L were concentrated 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 was diluted 30:70 with deionized water then injected onto 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 per 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 concentrations higher than 200 μg/L

were diluted 80:20 with methanol. 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). 
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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 (16 weeks of exposure ). There were eight replicate unexposed control tanks 

and four replicate tanks per prochloraz concentration. Each tank initially contained 30 embryos. Seven

stage 62 tadpoles per tank were randomly sub-sampled during weeks 5 – 7 of exposure, each tank was 

culled down to 10 animals per tank on week 9, and the remaining animals were sampled at 16 weeks of 

exposure. Prochloraz concentrations were chosen for reproductive effects based on studies using Rana 

temporaria (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 not shown).

2.5. Larval and Juvenile Necropsies

Sampling for the larval necropsy took place during weeks 5 - 7 of exposure. On the first day that an NF 

stage 62 froglet was seen (day 38 of exposure) the number of NF stage 62 froglets was counted in each 

tank. A random number from 1 to 6 was generated by throwing a die, then that number of stage 62

froglets was randomly taken from each tank for euthanasia and necropsy. This was repeated each day 

until seven froglets per tank had 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 to 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 buffered 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 the animals in each tank had completed 

metamorphosis. At week 8 of exposure, 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 euthanasia of the excess froglets.

Sampling for the juvenile necropsy 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 anesthesia was reached. The frogs were weighed and body length 

from snout to vent was measured. Blood was collected in heparinized MicrotainersTM (BD Biosciences, 

Franklin Lakes, NJ) by foot amputation and the foot was stored at -20°C for later genomic DNA 

extraction. Sex phenotype was determined by gross examination of gonads and the liver was removed 

and weighed. The forelimbs were examined for the presence of nuptial pads under 10X magnification. 
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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 centrifuged for 5 minutes at 10,000 g at 4°C, and the plasma separated and 

stored at -80°C until 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 

examined 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 previously 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 diluted 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 and standards were 

incubated with primary antibody and added to the plate in duplicate. Goat anti-rabbit IgG conjugated 

with horseradish peroxidase (Biorad) was used as a secondary antibody at a 1:16,000 dilution. Plates 

were incubated with tetramethylbenzidine substrate (TMB; Sigma) and the reaction was stopped after 

20 min with 1N 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 standard 

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 as 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 standard 

was made by dissolving 25 mg L-thyroxine (Sigma) in 25 mL 0.1 N sodium hydroxide and working 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 room 
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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 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 as zero if within the 95% CI of specific binding (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 paraffin embedding, sectioning, hematoxylin and eosin (H&E) staining, 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 storage in 10% NBF.

2.9. Statistics

Data were calculated from the means of eight replicate tanks for controls and four replicate tanks for 

exposed groups and analyzed using ANOVA with Dunnett’s t-test, Tamhane-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 to 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. Analytical 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 concentrations 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 μg/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 were 
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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

Percent spontaneous mortality in all tanks was 17.5%, 16.7%, 21.7%,7.5%, and 10.0% 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. Total 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 were no spontaneous deaths after 56 days of exposure. A Kaplan-Meier log rank 

analysis was performed, treating accidental deaths and euthanasia deaths for interim and final 

necropsies as censored data (Fig. 1A, B). There was no significant difference between control and 

prochloraz exposed groups, indicating that there was no significant mortality associated with prochloraz

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

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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 length from snout to 

vent. Larval lengths and weights for both male and female froglets in prochloraz exposed groups were 

similar to weights in unexposed controls (Fig. 2A, B). Juvenile lengths and weights for both male and 

female frogs were significantly decreased in the 180 μg/L prochloraz group compared to the controls 

(Fig. 2C, 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 groups, and significantly decreased in females from the 20 

μg/L group, compared to the controls (Fig. 3). Juvenile frogs in all groups did not display species 

characteristic sexual dimorphism in body size, probably due to immaturity. These data indicate that 

prochloraz exposure had no effect on body growth, sexual dimorphism, or size at 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 80 100 120

Survival

0.0

0.2

0.4

0.6

0.8

1.0

60 μg/L

6.7 μg/L

20 μg/L

180 μg/L

Control

Prochloraz μg/L

0 6.7 20 60 180

Percent Mortality

0

5

10

15

20

25

A B
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Figure 2. Body Size. Larval body weights (A) and lengths (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). Male () 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

1.0

Body Weight (g)

0

5

10

15

20

25

Prochloraz μg/L

0 6.7 20 60 180

Body Length (mm)

0

5

10

15

20

Prochloraz μg/L

0 6.7 20 60 180

Body Length (mm)

0

20

40

60

A

B

C

D

* *

* *

Prochloraz μg/L

0 6.7 20 60 180

Liver Somatic Index

0

1

2

3

4

5

6

7

*

* *

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

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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 41 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 prochloraz 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 determined for both larval and juvenile necropsies by gross examination of the 

gonad. Sex genotype was determined for both larval and juvenile 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 

determination of sex phenotype (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 juvenile 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. 7A, B). These data indicate that prochloraz

exposure had no effect on sex differentiation or juvenile sex phenotype.

Prochloraz μg/L

0 6.7 20 60 180

Days of Exposure

0

10

20

30

40

50

60
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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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 all 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 μg/L

0 6.7 20 60 180

Percent Female

0

20

40

60

80

100

Prochloraz μg/L

0 6.7 20 60 180

M/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 Female
USACEHR Technical 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, tubular 

eosinophilic droplets, and asynchronous 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 exposure, 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 

between cells within spermatocysts. The controls were minimally affected, and in the 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 (spermatocytes, 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 unexposed, 

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 mononuclear cellular infiltrate, 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 the 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 hypertrophy/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 frogs, 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 exposed 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 Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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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 droplet 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 spermatocytes (green 

arrow), late spermatocytes (yellow arrow), and developing spermatozoa (red arrow).

A B

C D
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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, 

mononuclear 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 with 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 infiltrates 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 μg/L

0 6.7 20 60 180

Percent

0

20

40

60

80

100

120

140

*

*

**

* *

*

*

Prochloraz μg/L

0 6.7 20 60 180

A B
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Figure 10. Ovary histopathology. Control ovary at 20X (A) showing diplotene oocytes separated by a 

single layer of flattened perifollicular 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

C
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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 histopathology. 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, pyknosis, karyomegaly, 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 controls

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 increased follicular lumen area in a few 

individuals. The increased incidence of follicular 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

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

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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 graded for hepatocellular atrophy and biliary epithelial hyperplasia. A 

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

hepatocellular atrophy was a common finding in prochloraz exposed frogs occurring in 21, 67, and 100% 

of 20, 60, and 180 μg/L prochloraz exposed frogs, respectively. Atrophy was not 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 mild 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 locations. 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 D
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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 atrophy. 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 intracytoplasmic 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 exposed 

frogs, respectively. Minimal intratubular accumulations of protein in scattered renal tubules were a 

consistent finding across all groups without concentration dependent correlation; 31, 33, 22, 25, and 

A B

C D
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38% of unexposed, 6.7, 20, 60, and 180 μg/L exposed frogs, respectively. Minimal vacuolization of renal 

tubules cells was observed 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 there 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 exposed 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 significantly 

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. An 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 interstitial 

fibrosis. Kidney at 40X from a 180 μg/L prochloraz exposed frog (D) showing intracytoplasmic 

amphophilic inclusions (red arrows).

A B

C D
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

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Figure 15. Non-gonad histopathology 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 were 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 protein ( ), 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 plasma vitellogenin levels (Fig 16A). In the juvenile sample, plasma VTG for females 

in the 180 μg/L prochloraz group was significantly increased compared to control females (Fig. 16C), 

p≤0.05. Due to the immaturity of the juvenile frogs, the VTG levels were very low compared to VTG 

levels in previously reported 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 μg/L

0 6.7 20 60 180

Percent

0

20

40

60

80

Prochloraz μg/L

0 6.7 20 60 180

A B

Prochloraz μg/L

0 6.7 20 60 180

Percent

0

20

40

60

80

100 C

ns ns

*

*

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

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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 groups for plasma T4 levels (Fig 16B). The juvenile sample showed no significant 

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.

Figure 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. There was no difference in body size 

between the control and prochloraz exposed groups in 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 (BrandePlasma T4

Prochloraz μg/L

0 6.7 20 60 180

T4 (ng/ml)

0

50

100

150

200

250

Prochloraz μg/L

0 6.7 20 60 180

VTG (mg/ml)

0.000

0.001

0.002

0.003

0.004

Prochloraz μg/L

0 6.7 20 60 180

T4 (ng/ml)

0

10

20

30

40

VTG mg/ml

0.000

0.005

0.010

0.015

0.020

A

B

C

D

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

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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, several 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 utero (Noriega, Ostby et al. 2005). 

Atrophy and biliary epithelial hyperplasia were the primary histopathological findings in the livers of 

juvenile frogs exposed to high concentration prochloraz. While prochloraz generally inhibits 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 stress biomarkers in stickleback (Sanchez, Piccini et al. 2008). Additionally, 

ketoconazol, 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 intracytoplasmicamphophilic 

inclusions within the renal tubular epithelium of high dose juvenile frogs which are reminiscent of 

Mallory bodies seen in alcoholic liver 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 perturbation of the hypothalamicpituitary-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. Plasma 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 ng/mL 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 (Miyauchi, LaRochelle, Jr. et al. 1977), compared to 124 ng/mL 
USACEHR Technical 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 dilution but not 

eliminated. For this reason, it is possible that plasma T4 levels determined in this study may be 

artificially 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, nonspecific, general 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 changes 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 different 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 reflect a relative increase due the 

loss of spermatocytes/spermatids rather than an absolute increase in numbers.

The degenerative testicular changes in juvenile X. laevis in this study were 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 regression in males (Kelly, 1996) which is similar to the feminization of the 

secondary sexual characteristics of male rats during perinatal prochloraz 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 fibrosis 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 females and delay in ovarian development (Kinnberg, Holbech et al. 2007). The minimal 

effect on the ovary compared to the testis may be related to the additive effects of androgen 

antagonism and suppression of steroidogenesis on the more androgen sensitive testes.

In this study, VTG, an estrogen inducible yolk protein, was significantly increased in 180 μg/L prochloraz 

exposed juvenile females, which was unexpected. In other studies, adult rainbow trout exposed to 100 
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

25

μg/L prochloraz had no decrease in VTG (Le, Thomas et al. 2001), adult male zebrafish exposed to 16 

and 64 μg/L prochloraz showed a significant increase in VTG, but 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 (OECD, 2006). However, adult 

fathead minnows exposed to 20 - 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 and Conclusions

Besides specifically determining the effects of prochloraz exposure on X. laevis, the general intent 

behind this study was to validate a tier II amphibian assay for endocrine disruptors. Thus, practical 

considerations of time and resources figured 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 linked 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 at 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 

radioisotopes or LC–ICP–MS (Simon, Tietge et al. 2002). Additionally, the selection of X. laevis 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 exposed to high concentration prochloraz exhibited mild thyroid pathology 

and juveniles exhibited mild ovarian pathology, mild to moderate testicular pathology, and increased 

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 assistance with statistical 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 commercial 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 other 
USACEHR Technical Report: Effects of Prochloraz Exposure on Larval and Juvenile Xenopus Laevis

26

Federal 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 Assessment and Accreditation of Laboratory Animal Care, 

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

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List of Abbreviations and Acronyms

AR androgen receptor

°C degree Celsius

CaCO3 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 tricaine methanesulfonate

NBF normal buffered formalin

ng nanogram
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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 hormone

USACEHR U.S. Army Center for Environmental Health Research

US EPA U.S. Environmental Protection Agency

VTG vitellogenin
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Appendix A

Data Summary Tables
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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 ± 0.06 0.72 ± 0.17 0.59 ± 0.12 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 52.7 ± 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 ± 0.29 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 Pads 2.8 ± 6.8 12.5 ± 16 15.1 ± 14 7.1 ± 14 12.5 ± 25

Female Percent Nuptial Pads 0.0 0.0 0.0 0.0 4.2 ± 8.3
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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)

Alla 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.
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Appendix B

Histopathology Scoring Tables
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Table 1. Larval thyroid histopathology scoring.

Nominal Prochloraz Concentration (μg/L)

0 6.7 20 60 180

Thyroid Gland Hypertrophy

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 ns ns 7.1 ± 8.2*

Follicular Cell Hypertrophy

Percent Mild 7.1 ± 14 14.3 ± 11 ns ns 48 ± 18*

Follicular Cell Hyperplasia

Percent Mild 3.6 ± 7.1 14.3 ± 11 ns ns 48 ± 26

Increased Follicular Lumen

Percent Mild 0.0 ± 0.0 0.0 ± 0.0 ns ns 7.1 ± 8.2*

ns = not scored

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

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 ± 37
USACEHR Technical 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

Gonadal 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 ± 33

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 Technical 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

Gonadal 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 Infiltrate

Percent All Grades 60 ± 27 79 ± 25 45 ± 39 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