Test Chemical: 17 β

- estradiol (E2)

MOA: estrogen

Protocol: full MMT

Lab: CERI (Japan)

Summary of Test Chemical and MOA:

Obviously, E2 is a natural estrogen hormone that also has been found in the environment at ng/L

concentrations and has been shown to induce feminization in many different forms across various species.

Because of this, E2 is an excellent model EDC to test the validity of an EDC

- related bioassay such as the

MMT.

Summary of Protocol:

The MMT completed by CERI t esting the effects of E2 was done with a full research MMT

protocol

. Briefly, the E2 MMT protocol spanned 2 1⁄2 generations, in which in the F0 generation chemical

exposure was initiated with adult spawning pairs and continues through the end of the reproductive

assessment . In the F1 and F2 generations, fish were continuously exposed from embryo through adult

spawning. Fish were sampled at two major life

- stages; subadults (approximately 8 wpf) in the F1 and F2

generations, and mature adults ( approximately 16 wpf) in F0, F1, and F2. Reproduction was evaluated

for 5 consecutive days per week over 4 weeks starting at 10 wpf

The general design employed 6 treatments including dilution water controls and 5 trea tment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The

proportionate chemical concentration diffe rence between treatments was approximately 0.3. The average

measured E2 concentrations in the test were 0.9

, 2.8

, 8.9

, 28 , and 84 n g/L. Reproduction in all three

generations (F0, F1, and F2) consisted, nominally, of 6 breeding pairs per concentration and 6 breeding

pairs for controls. These breeding pairs were also the fish sampled as adults.

Summa ry of Results (Table 1) :

The LOECs are summar ized in a “heat table” with both the direction of the effect, and the

statistical test used indicated. The LOEC is color

- coded with more intense colors for lower

concentrations of E2

. There were no viable fish available in the 84 ng/L treatment of F2, so only effects

at 28 n g/L or lower were observable. The LOEC for fecundity and fertility, arguably the most important

endpoints were 28 ng/L. The LOECs across the entire E2 MMT ranged from 2.8 to 84 ng/L, seemingly

centered around 28 ng/L, and predominately affected XY fish, as expected with an estrogenic EDC.

The suite of biological impacts on the fish within the MMT could be linked across levels of

biological organization to the we ll documented mode of action of E2. For instance at the

population/organismal level, fecundity, was reduced starting at 28 ng/L in F1). At the individual/tissue

level of organization, feminized testes were observed in XY individuals exposed to as low as 28 ng/ L E2.

At the same time and further following an estrogenic mode of action now at the molecular level, 4tOP

induced vitellogenin gene expression in XY fish as low as 28 ng/L. So again all the observed effects

within the MMT “passed muster” as attrib utable to E2 binding to and activating the estrogen receptor.

Reproduction (Figure 1) :In F0, E2 did not reduce either fecundity or fertility during the exposure/reproductive assessment

period. Even when the reproductive output was analyzed by week and E2 exposure, fecundity and

fertility remained the same as controls.

In F1, the LOEC for both reduced fecundity and fertility was 28 ng/L E2. The fecundity was

reduced from 26.7 eggs per pair in controls to 18.6 and 6.8 eggs per pair in the 28 and 84 ng/L treatments.

Likewise, the fertility was reduced from 25.9 (97%) fertile eggs per pair in controls to 6.4 (34%) and 0.9

(13%) fertile eggs per pair in the 28 and 84 ng/L treatments.

Furthermore, there were unexplained differences between the week ly egg outputs within

treatments including control. This observation is difficult to reconcile with the study design. For

instance, there is no obvious reason why one week of control output should be different than any other

week of control output in the F1 generation.

In F2, the LOEC for reduced fertility only was 28 ng/L while there was not an observed LOEC

for reduced fecundity. The fertility was reduced from 18.0 (85%) fertile eggs per pair in controls to 12.7

(78%) fertile eggs by exposure to 28 ng/L E2. This is a more intermediate response to E2 exposure than

was observed at the same concentration in the F1 reproductive assessment. Again there were some

unexplained differences between weekly egg outputs within treatments including the contro ls.

Adult Vitellogenin (Figure 2) :

In all three generations, the LOEC for increased vitellogenin production in the liver of males was

28 ng/L E2. At 84 ng/L E2 in F0 and F1 (there were no fish in this treatment in F2), the males had over

5 00 times mo re vitellogenin detected than the control males. At 28 ng/L in all three generations, the

increased vitellogenin observed was, while more modest, still significantly increased from the control

mean by about 38 times in F0 and F2, and by about 80 times in F1. A s anticipated there was no effect on

vitellogenin production in adult XX fish through the E2 MMT

.

Adult Secondary Sex Character s (Figure 3) :

There was no change in the number of papillae in the F0 adult males after exposure to E2. This is

a typical result for F0, as the papillae had already developed in male fish by the time exposure began , and

the process of resorption seems to require a longer period of time, be yond the 5 week exposure of F0

.

However, in F1, the E2 exposure did reduce the number of papillae observed in males exposed to

either 28 or 84 ng/L. At the LOEC of 28 ng/L, the mean number of papillae per XY fish was 67 as

compared to 117 in the XY control fish. In addition, none of the XY fish in the 84 ng/L treatment had a

singl e papilla.

In F2, there were no statistically significant changes in SSC. However, the mean number of

papillae for XY fish in the 28 ng/L treatment (the highest concentration present in F2) was, in essence, the

same as seen in F1: 71 versus 67. Howev er, the control mean count was only 86, much lower than the

117 that was seen in F1. While impossible to conclude that there was a reduction at 28 ng/L E2 in F2, the

results are suggestive of similar sensitivity between the two generations.

Adult Gonad phenotype (Figure 4) :

In the adult XY fish of both F1 and F2, E2 altered the gonad phenotype starting at the LOEC of

28 ng/L. At this concentration, there were significant numbers of medaka with intersex testes. Evenmore dramatically, 84 ng/L induced the complete feminization of the XY gonads of all the adults of the

treatment.

Adult Pathology (no figure) :

No pathology was recorded for adult fish in this MMT.

Adult Weight (no figure ) :

No weight data was collected in adult fish in this MMT.

Adult Length (no figure) :

No weight data was collected in adult fish in this MMT.

Adult Survival (no figure) :

No weight data was reported in adult fish in this MMT.

Subadult Vitellogenin (Figure 5) :

In subadult medaka, E2 caused significant increases in hepatic vitellogenin in both XX and XY

fish across both F1 and F2 generations. In F1, the LOEC was 84 ng/L for XX fish and 28 ng/L for XY

fish. Specifically, the vitellogenin increased from 1197 ng/mg liver in the control XX fish to a mean of

3998 ng/mg liver in the 84 ng/L treatment. In addition at 28 ng/L of E2, the XX medaka had, what

appeared to be, elevated vitellogenin (2165 ng/mg liver) even though it was not statistically significant.

This level of hepatic vitellogenin is equivalent to that found in the corresponding XX fish of F2 (see

be low). In XY fish, the concentration of vitellogenin increased from 1.2 ng/mg liver (with many of the

samples below the detection limit) to 462 ng/mg liver and 4039 ng/mg liver in the 28 and 84 ng/L

treatments, respectively.

The LOEC in F2 subadults wa s 8.9 ng/L in XX medaka and 28 ng/L in XY medaka. The XX

mean in the 8.9 ng/L treatment and the 28 ng/L treatment was 2238 ng/mg liver and 2213 ng/mg liver,

respectively, as compared to 979 ng/mg liver in the control XX fish. This increase in 28 ng/L tre atment

was similar to what was observed at the same does in F1. The individual XY control vitellogenin

measurements were almost all below the detection limit (the calculated mean was 0.4 ng/mg liver) while

the 28 ng/L E2 treatment group had a mean of 762 ng/mg liver, which was statistically the same as seen

in F1 in this treatment.

Subadult Secondary Sex Characters (Figure 6) :

In F1, the LOEC of E2 decreasing the number of anal fin papillae in XY fish was 2.8 ng/L while

the LOEC in F2 was 28 ng/L. Howe ver, the means across both generations were very similar throughout

the various treatment concentrations. So while the intergenerational LOECs were very different, the

mean number of papillae from corresponding treatments were very similar. For instance, the mean

number of papillae on XY fish exposed to 8.9 ng/L E2 in F1 was 57 (this was significantly lower than the

F1 control mean) while it was 60 in F2 (this was not significantly lower than the F2 control mean), and

furthermore, the mean from the F1 8.9 ng/L E2 group was not statistically different (in other words they

are the same) than the mean from the F2 8.9 ng/L E2 group.

Subadult Gonad Phenotype (Figure 7 ) :In F1 s ubadults XY fish, E2 induced significant changes in the gonad phenotype starting at 28

ng/L and increasing in magnitude in the 84 ng/L treatment. At the LOEC (28 ng/L), there was a single

XY individual out of 12 that had completely feminized gonads and 3 of 12 had intersex gonads.

Increased E2 concentration to 84 ng/L resulted in 11 of 12 XY fish having completely feminized gonads.

Similarly, an elevated number of F2 XY fish exposed 28 ng/L E2 had feminized gonads: 1 fish with

complete ovaries and 4 of 12 with intersex gonads.

Suba dult Pathology (no figure) :

No pathology was recorded for adult fish in this MMT.

Suba dult Weight (Figure 8) :

Data was not presented with genotypic or phenotypic sex information so subadult weight is

presented sans gender.

E2 did not alter weight in F1 subadult fish, however a slight , but statis tically significant, increase

from a mean of 208 mg in controls to 227 mg in the 28 ng/L treatment (the highest concentration present

in F2) of F2 was recorded.

Suba dult Length (Figure 9) :

Data was not presented with genotypic or phenotypic sex informat ion so subadult length is

presented sans gender.

E2 did not alter length in F1 subadult fish, however a slight, but statistically significant, increase

from a mean of 2.7 mm in controls to 2.8 mm in the 28 ng/L treatment (the highest concentration present

in F2) of F2 was recorded.

Subadult Survival (no figure ) :

There was not a treatment

- related increase in mortality associated with E2 exposure reported for

either generation.

Hatch (Table 2) :

T he hatch of F1 was not lower by E2 exposure. However, the hatch of F2 embryos was lowered

in the highest treatment present, 28 ng/L, from the control mean of 96% to 53%.E2 MMT LOEC Summary E2 (ng/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

subadult subadult adult adult adult 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 0.9 0.11

Weight LOEC>84 28↑ 3 2.8 0.35

Length LOEC>84 28↑ 4 8.9 1.04

Survival LOEC>84 LOEC>84 5 28 2.9

Hatch LOEC>84 28↓ 6 84 7.1

Papillae 2.8 ↓ 28↓ LOEC>84 28↓ LOEC>28

V tg 28 ↑ 84 ↑ 28 ↑ 8.9 ↑ 28↑ LOEC>84 28↑ LOEC>84 28↑ LOEC>28

Gonad Phenotype 28 ↑ LOEC>84 28 ↑ LO EC>28 28↑ LOEC>84 28↑ LOEC>28

Fecundity LOEC>84 28↓ LOEC>28

Fertility LOEC>84 28↓ 28↓

Highest Conc 84 28 84 84 28. Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation,

and the whisker is the standard error of the number of eggs spawned per

day by each replicate. * = significantly different from control. E2 F0 Reproduction CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility E2 F1 Reproduction CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility



# * *

* * E2 F2 Reproduction CERI MMT

0 0.9 2.8 8.9 28

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility



# *Figure 2, adult vitellogenin. Gender specific hepatic vitellogenin of adult

medaka in F1 (upper left), F2 (upper right), and F0 (lower left) generations.

The central line is the mean, the box is the standard deviation and the

whisker is the standard erro r of the vitellogenin expression. Genotypic sex

was determined in F1 and F2, and phenotype was used for gender in F0. * =

significantly different from gender specific controls. E2 F2 Adult Vitellogenin CERI MMT

Concentration (ng/L)Vtg Protein (ng/mg liver)

0 0.9 2.8 8.9 28 0 1000 2000 3000 4000 5000 6000

XY

XX



## *

E2 F0 Adult Vitellogenin CERI MMT

Concentration (ng/L)Vtg Protein (ng/mg liver)

0 0.9 2.8 8.9 28 84 0 1000 2000 3000 4000 5000 6000

Male

Female



## * * E2 F1 Adult Vitellogenin CERI MMT

Concentration (ng/L)Vtg Protein (ng/mg liver)

0 0.9 2.8 8.9 28 84 0 1000 2000 3000 4000 5000 6000

XY

XX



## *

*Figure 3, adult secondary sex characters. Male secondary sex characters

(SSC) of adult medaka in F1 (upper left), F2 (upper right), and F0 (lower left)

generations after exposure to E2. The central line is the mean, the box is

the standard deviation and the whisker is the standard error of the nu mber

of anal fin papillae, that is the SSC. Genotypic sex was determined in F1 and

F2, and phenotype was used for gender in F0. * = significantly different

from controls. E2 F1 Adult XY SSC CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0 20 40 60 80 100 120 140 160 180Anal Fin Papillae (#/fin)



## *

* E2 F2 Adult XY SSC CERI MMT

0 0.9 2.8 8.9 28

Concentration (ng/L) 0 20 40 60 80 100 120 140 160Anal Fin Papillae (#/fin)

E2 F0 Adult Male SSC CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0 20 40 60 80 100 120 140 160Anal Fin Papillae (#/fin)Figure 4, adult gonad phenotype. Gonad phenotype of XY adult medaka in F1 (upper) and F2 (lower) generations after

exposure to E2. The stacked bar graphs show the percent of individuals with testis, intersex, or ovary. The number in

the boxes is the number of individuals in each treatment with that gonad phenotype. Intersex gonad is defined as a

single gonad with any amount of both ovarian and testicular tissue with no measurement of relative proportion.Figure 5 , subadult vitellogenin. Gender specific vitellogenin of subadult medaka in F1 (upper) and F2 (lower)

generations. The central line is the mean, the box is the standard deviation and the whisker is the standard error of the

vitellogenin expression. E2 F1 Subadult Vitellogenin CERI MMT

Concentration (ng/L)Vtg Protein (ng/mg liver)

0 0.9 2.8 8.9 28 84 0 1000 2000 3000 4000 5000 6000

XY

XX



# * * *

E2 F2 Subadult Vitellogenin CERI MMT

Concentration (ng/L)Vtg Protein (ng/mg liver)

0 0.9 2.8 8.9 28 0 1000 2000 3000 4000 5000 6000

XY

XX



# * *

*Figure 6 , subadult secondary sex characters. Male (XY) secondary sex characters (SSC) of sub adult medaka in F1

(upper) and F2 (lower ) generations after exposure to E2 . The central line is the mean, the box is the standard deviation

and the whisker is the standard error of the number of anal fin papillae that is the SSC

. E2 F1 Subadult XY SSC CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0 20 40 60 80 100Anal Fin Papillae (#/fin)



# * * *

*

E2 F2 Subadult XY SSC CERI MMT

0 0.9 2.8 8.9 28

Concentration (ng/L) 0 20 40 60 80 100Anal Fin Papillae (#/fin)



# *Figure 7, subadult gonad phenotype. Gonad phenotype of XY subadult medaka in F1 (upper) and F2 (lower) after

exposure to E2 . The stacked bar graphs show the percent of individuals with testis, intersex, or ovary. The number in the

boxes is the number of individuals in each treatment wit h that gonad phenotyp e . Intersex gonad is defined as a single

gonad with any amount of both ovarian and testicular tissue with no measurement of relative proportion.Figure 8, subadult weight. Weight of sub adult medaka in F1 (upper) and F2 (lower ) generations after exposure to E2

. The central line is the mean, the box is the standard deviation and the whisker is the standard error of the weight

. DMY

information was not collected so data is not categorized by gender. E2 F1 Subadult Weight CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40Weight (mg)

E2 F2 Subadult Weight CERI MMT

0 0.9 2.8 8.9 28

Concentration (ng/L) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40Weight (mg) 

# *Figure 9, subadult length. Length of sub adult medaka in F1 (upper) and F2 (lower ) generations after exposure to E2

. The central line is the mean, the box is the standard deviation and the whisker is the standard error of the weight

. DMY

information was not collected so data is not categorized by gender. E2 F1 Subadult Length CERI MMT

0 0.9 2.8 8.9 28 84

Concentration (ng/L) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Length (mm)

E2 F2 Subadult Length CERI MMT

0 0.9 2.8 8.9 28

Concentration (ng/L) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Length (mm) 

# *Table 2. Hatch

Hatch (mean % )

Treatment

Generation control 0.9 n g/L 2.8 n g/L 8.9 n g/L 28 n g/L 84 n g/L

F1 96 99 92 95 91 97

F2 96 85 97 86 53 XTest Chemical: 4

- tert

- octylphenol (4tOP)

MOA: estrogen (estrogen receptor agonist)

Protocol: full research MMT

Lab: MED

Summary of Test Chemical and MOA:

4tOP is an active degradation product of octylphenol ethoxylates , which are widely used

nonionic surfactants used in the manufacture of cleaning agents, plastics, paper, cosmetics and food

products. 4tOP has been shown to be estrogenic in both in vitro systems and in fish exposures including

me daka. For instance in a 60 day exposure starting with fertilized medaka eggs, 4tOP induced hepatic

vitellogenin production and altered sexual differentiation (intersex gonads) at a LOEC of 11.4 μ g/L.

Needless to say, the estrogenic properties of 4tOP are well documented as well as its presence in many

aquatic environments.

Summary of Protocol:

The MMT completed by USEPA

- MED t esting the effects of 4tOP was done with the full research

MMT protocol that has been detailed previously (pp. ). Briefly, the 4tOP MMT protocol spanned 2 1⁄2

generations, in which in the F0 generation chemical exposure was initiated with spawning pairs at the

beginning of 1 0 wpf (69 dpf) and continues through the beginning o f 15 wpf (105 dpf) at which time the

fish were sacrificed. In the F1 and F2 generations, fish were continuously exposed from embryo through

adult spawning. Fish were sampled at two major life

- stages; subadults (in the 7 th wpf ; 44

- 45 dpf) in the

F1 and F2 generations, and mature adults (at the start of the 15 th wpf ; 105 dpf ) in F0, F1, and F2.

Reproduction was evaluated for 4 consecutive days per week over 5 weeks from 10 wpf to 15 wpf.

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The

proportionate chemical concentration diffe rence between treatments was 0.5. The average measured 4tOP

concentrations in the test were 6, 13, 26, 51, and 102 μ g/L. Reproduction in all three generations (F0, F1,

and F2) consisted, nominally, of 6 breeding pairs per concentration and 6 breeding pairs for controls.

These breeding pairs were also the fish sampled as adults. At the subadult (7 wpf; 44

- 45 dpf) sampling, 8

of the 10 fish per aquaria were randomly sacrificed to a) produce the subadult samples and to b) leave

behind the breeding pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair (4

consecutive days per week over 5 weeks) in F0, F1, and F2; in adults in F0, F1, and F2, length, weight,

VTG gene expression, SSC, and histopathology evaluation of gonad, kidney, and liver were recorded; and

in subadults in F1 and F2, VTG gene expression, SSC, gonad histolo gy, weight and length were recorded.

In addition, hatch and survival from hatch to 3 wpf, 3 wpf to 7 wpf, and 8 wpf to 15 wpf were recorded.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with both the direction of the effect, and the

statistical test used indicated. The LOEC is color

- coded with more intense colors for lower

concentrations of 4tOP

. In general, the F0 generation is less sensitive than the F1. There were no viable

fish available in the 102 μ g/L treatment of F2 , so only effects at 51 μ g/L or lower were observable. In

addition, the LOECs obtained at the subadult samplings were lower than those of the adult samplings.

However, whether this was a result of a greater sensitivity at this lifestage or a result of th e statistical

impact of greater sample size per replicate at the subadult sampling is difficult to determine from the data.The LOEC for fecundity and fertility, arguably the most important endpoints were 102 μ g/L and 51 μ g/L

in F1 and F2, respectively. The LOECs across the entire 4tOP MMT ranged from 13 to 102 μ g/L,

seemingly centered around 51 μ g/L, and predominately affected XY fish, as expected with an estrogenic

EDC.

The suite of biological impacts on the fish within the MMT could be linked across levels of

biological organization to the well documented mode of action of 4tOP as an ER agonist. For instance at

the population/organismal level, fecundity, was reduced starting at 102 μ g/L in F1), and probably 51 μ g/L

in F2. At the individual/tissue le vel of organization, feminized testes were observed in XY individuals

exposed to as low as 13 μ g/L 4tOP, but maybe more typically at 25 μ g/L. At the same time and further

following an estrogenic mode of action now at the molecular level, 4tOP induced vite llogenin gene

expression in XY fish as low as 25 μ g/L. So again all the observed effects “passed muster” as attributable

to 4tOP binding to and activating the estrogen receptor.

Reproduction (Figure 1) :

In F0, there is no single week with an affect s een from 4tOP exposure on either fecundity or

fertility across all treatment levels. While there was not a decrease in either fecundity or fertility through

time, there does seem to be a trend, albeit a non

- significant one, toward lower fecundity and fertility with

increasing concentration of 4tOP. The magnitude of the drop in both fecundity and fertility from controls

to the significant treatments was fairly small, and all treatment groups produced enough fertile eggs to

continue into F1.

It came as a surprise that the inclusion of time into the statistical model for F0 reproduction did

not strengthen the model. Because these are F0 fish that had not been exposed to 4tOP prior to the start of

the MMT at adulthood, it was assumed that any d ecrease in either fecundity or fertility would be

exacerbated as the exposure continued through 5 weeks of reproductive assessment. It has been

postulated that any effect of 4tOP through time on reproduction was masked somewhat by a general

trend, across the entire F0 cohort, of increasing reproductive output through the 5 weeks of assessment,

presumably as a result of developmentally reaching prime spawning maturity toward the end of the

assessment period.

In F1, there was a statistically significan t increase over controls in both fecundity and fertility in

groups exposed to 6

– 25 μg/L 4tOP, which unfortunately seemed to be consequence of lower than

normal egg production in the controls. Also, it was expected that an increase in fertile eggs in any

exposure group would not be considered an adverse outcome, therefore, these affects have been generally

ignored in the analysis. The 102 μg/L treatment had lower fecundity and fertility than controls in all

weeks assessed, except in 13 wpf. In addition to being statistically significant, the magnitude of the

decrease was substantial and resulted in not enough embryos to continue this treatment group into F2.

Much of the time, the breeding pairs exposed to 102 μg/L 4tOP produced no eggs, and when they di d

produce eggs, the eggs usually were not fertile, which may indicate effects on both male and female fish.

In addition, the pairs exposed to 51 μg/L, while not statistically different from controls in F1, were lower

than what would be anticipated in norm al reproduction. Internal MED power analyse s indicate that the

MMT had low ability to detect decreases in fecundity of less the 50%, therefore it is not surprising to see

trends in the data that are not statistically significant.

In F2, there were no 102 μg/L exposed fish available for breeding pairs. While the 51 μg/L fish

trended lower than controls, the fecundity and fertility were not different in any particular week than

controls except at 11 wpf when the fertility was lower than controls. Taki ng the whole dataset (removing

time from the model), the 51 μg/L exposure group did have reduced fecundity when compared tocontrols. As already discussed, given the lower power in the 4tOP MMT, it is not surprising to have

significance in F2, 51 μg/L 4tO P that was not seen in F1 at the same concentration even though the

general trends and character of the data from F1 and F2 are quite similar.

Adult Vitellogenin (Figure 2) :

In the F0, F1, and F1 adult samples, the effect of 4tOP exposure on vitellogen in gene expression

had both similar trends of increased vitellogenin gene expression with increased concentration. In all

three generations starting at 51 μ g/L , there was a significant increase in vitellogenin mRNA in adult XY

fish. The number of copies of the gene produced by XY fish after exposure to 51 μ g/L was about the

same in the three generations: F0 = 4 x 10 5 , F1 = 5 x 10 5 , and F2 = 6 x 10 5 , even though the relative

magnitude of the effect (how many times more copies of the gene were detected in the exposure groups

relative to the controls) was not as similar across the generations: F0 = 60 times, F1 = 10 times, and F2 =

93 due to relatively high copy numbers detected in the control XY fish in F1. Regardless, the LOEC for

adult vitellogenin indu ction in XY fish was the same across all three generations, and as anticipated there

was no effect on vitellogenin mRNA copy number in XX fish through the 4tOP MMT.

Adult Secondary Sex Character s (Figure 3) :

In F0, 4tOP did not affect the number of anal fin papillae , the measure of secondary sex

characters in medaka, in XY fish. However in this generation, the papillae have already developed in

male fish by 10 wpf when the exposure begins, and the process of resorption seems to require high

concentr ations of estrogenic EDCs for prolonged periods of time that are beyond the 5 week exposure to

4tOP. In F1, XY fish in the 102 μg/L exposed treatment did not have any papillae (which is the female

phenotype) while the XY fish in the 51 μg/L group had fewe r, but not significantly fewer, than controls.

In addition, F2, XY fish in the 51 μg/L, 26 μg/L, and 13μg/L treatment groups had reduced numbers of

anal fin papillae as compared to controls. However, this may be more a reflection of higher than usual

pap illae counts in the F2 XY controls rather than a 4tOP induced decrease at 13 and 25 μg/L. On the

other hand, the number of papillae observed in the 51 μg/L group was certainly lower than typical

controls. Therefore, the F2 LOEC for SSC of 13 μg/L should be considered as tentative considering the

data for SSC from the entire 4tOP MMT.

Adult Gonad phenotype (Figure 4) :

In the adult samples (15 wpf) , intersex was not observed in any of the control XY fish of F0, F1

and F2 generations, but it was observed in low, but not statistically significant, prevalence (1 per

treatment) in all 4tOP treatments in F0, except 51 μg/L. In F1, the prevalence increased with treatment

level from two intersex XY fish in the 13 μg/L treatment to 4 in the 51 μg/L treatment. T he highest dose

(102 μg/L) had 2 of the 6 XY fish with intersex gonads and the remaining 4 XY fish had only ovaries. In

F2, 2 of 3 XY fish in the 51 μg/L treatment (the highest remaining treatment) were intersex while the last

remaining fish in the treatm ent was completely sex reversed. On the other hand, both the 13 and 25 μg/L

treatments each had a single XY fish with an intersex gonad.

The difference in sensitivity between F0 and F1/F2 is most likely a reflection of exposing F0

starting with adult s, while the F1/F2 fish were exposed for their entire lifecycle. Not only was the

exposure longer in F1/F2 than F0 adults, it also encompassed the more sensitive early stages of

development, and in the end, these inter

- generational differences result from comparing activational

effects to developmental effects. However, the different LOECs observed between F1 and F2 cannot be

explained in this manner. Of course, one explanation is that indeed F1 was more sensitive than F2.

While this could be explained by some selection process that resulted in more resistant animals spawningin F1 leading a less sensitive generation in F2, this seems unlikely, and in fact, the most probable

explanation is that the difference between F1 and F2 was an artifact of a small sample size

.

Adult Pathology (Tables 2

– 3) :

Multiple organs including gonad, liver, and kidney were analyzed by the pathologist for lesions

resulting from exposure to 4tOP that were both specific to an estrogenic mode of action or were indicative

of mo re generalized toxicity. Generally, the observed pathologies in F0 were of low severity with most of

the statistically significant pathologies having severity scores of 1, and the remainder having severity

scores of 2. The pathologies with severity score s of 1 in adult F0 XY fish were interstitial fibrosis and

proteinaceous fluid in the gonad, nephropathy in the kidney, and hyperplasia in the swim bladder, while

those with severity of scores of 2 were proteinaceous fluid in the kidney, and basophilia of t he liver. The

XX fish also had proteinaceous fluid in the kidney with a severity of 1, and in addition these fish had

cystic degeneration in the liver. All the observed pathologies occurred were statistically significantly

only after exposure to 102 μg/L 4tOP.

In adult F1 XY medaka, the many of the observed pathologies tended to be more severe than in

the F0 fish. Both nephropathy and proteinaceious fluid in the kidney occurred at the same concentration

(102 μg/L) and severity score (1) as in F0 an d consisted of glomerulomegaly, mineralization, dilatation,

and vacuolization of the renal tubules. As one would expect, testicular oocytes were significantly

increased over control XY fish down to 13 μg/L 4tOP (severity score of 1) and with highest sever ity score

of 4 occurring at 102 μg/L. In addition, the liver had diffuse hepatocellular basophilia at a severity score

of 2 and 3 at 51 and 102 μg/L, respectively. There were no statistically significant pathologies in F1 XX

medaka.

The F2 adult, XY pa thologies were only found in the gonad and the kidney. Gonad pathologies

included increased spermatogonia (1

- 2 at 25 and 51 μg/L), mononuclear infiltration (1 at 51 μg/L), and

asynchronous development (1

- 3 at 25 and 51 μg/L); kidney pathologies included g lomerulomegaly (3 at

51 μg/L), mineralization (1 at 6 μg/L), and hyperplasia of hematopoietic (1 at 51 μg/L). Only one

pathology was significant in adult F2 XX fish (Table 6 ): asynchronous development in the gonad (2 at 51

μg/L).

Adult Weight (Figure 5) :

The weight in adult medaka, in either XX or XY fish, was not decreased by any 4tOP treatment in

any of the generations. Weight was increased over controls in F0,XX fish by exposure to 102 μg/L 4tOP,

and in F1, weight was increased in XY fis h exposed to 51 and 102 μg/L 4tOP. There were no affects on

weight in F2. All of these effects were relatively small in magnitude, and were not anticipated to be a

negative consequence of exposure.

Adult Length (Figure 6) :

After 102 μg/L exposure to 4tOP, F0 XX fish were slightly longer than controls, however this

trend did not continue across the other generations. There were no effects on length in XY F0 and F2

fish; while in F1, the XY fish in 13 μg/L through 102 μg/L 4t OP exposed fish were slightly longer than

controls. This actual increase was small in absolute terms, and the Jonckheere

- Terpstra trend test that

detected the increase over controls was by

- and

- large influenced by much longer fish in the 13 and 51

μg/L tre atments.

Adult Survival (Table 7) :No effects were seen after 4tOP exposure to the survival of adult medaka from 8 to 15 wpf of

either gender.

Subadult Vitellogenin (Figure 7) :

In subadult fish, 4tOP caused significant increases in vitellogenin gen e expression of XY fish in

the 25 μg/L through 102 μg/L treatment groups of the F1 generation. The level of gene induction was

approximately 6 times greater copy number with exposure to 25 μg/L 4tOP than controls, and the level of

induction increased to a bout 200 times more than controls in the upper two treatments. In addition to the

effects on XY medaka, 4tOP exposure also increased vitellogenin gene expression in XX individuals in

the 51 and 102 μg/L treatments by a factor of 3

- 4 times.

A similar t rend is also evident in the subadults of the F2 generation. The LOEC in the XY fish

was again 25 μg/L 4tOP where the level of induction was about 5 times that of controls while in the next

treatment higher (51 μg/L), the level of induction was about 100 t imes that of controls. Like the F1

generation, there was a slight increase in vitellogenin gene expression (~4 times) at 51 μg/L in XX fish in

the F2s.

Subadult Secondary Sex Characters (Figure 8) :

At 7 wpf, the secondary sex are not fully developed an d some XY individuals have not yet

displayed any papillae at the time of sacrifice so the variance tends to be high. This makes SSC a less

definitive measure of gender phenotype at the subadult stage than it is in adults. In F1 XY fish sampled

as subadul ts, the anal fin papillae were reduced in the 102 through 25 μg/L treatments when compared to

controls, and paradoxically, anal fin papillae in the 13 μg/L treatment were statistically higher than

controls. Given the mode of action of 4tOP, the rather low number of papillae in controls in F1 and the

results in F2, this seems most likely to be an anomalous finding. In F2 XY medaka, the number of

papillae was reduced in 51, 25, and 13 μg/L, when compared to controls.

The general trends of the effect of 4tOP exposure are similar between F1 and F2. There was a

difference in the LOEC with the F2 LOEC (13 μg/L) lower than the F1 LOEC (25 μg/L) by a single

treatment level. There are a two factors, other than a presumable difference in response between F1 an d

F2, that may have contributed to the difference: 1) the mean control value for both F1 and F2, the F1

control mean is less than would be expected while the F2 control value is higher than would be expected,

and 2) an oddly high mean in the F1, 13 μg/L tr eatment. These two factors seemed to have contributed to

measuring a LOEC of 25 μg/L in F1 and 13 μg/L in F2.

Subadult Gonad Phenotype (Figure 9) :

In F1 subadults, 4tOP lead to increased levels of intersex gonads in XY medaka at 25 μg/L and

above. There were a small number of intersex gonads observed in the control XY fish, however these

instances were characterized by very few, small undeveloped oocytes, while the severity of the observed

intersex gonads seen in the statistically significant concen trations tended to be more extreme. Along with

intersex gonads, XY medaka with ovarian gonads were observed , starting at 25 μg/L (not statistically

significant) with 1 case or 4% of the treatment, increasing to 11 cases or 38% of the XY fish in the 51

μ g/L treatment, and finishing with 100% of the 5 XY fish in the 102 μg/L treatment.

In F2, a similar pattern of gonad phenotypes was observed in the XY subadult fish: statistically

elevated numbers of interex fish in both the 25 and 51 μg/L treatments: 6 and 9, respectively (no fish in

the 102 μg/L treatment). In addition, sex reversal w as observed in both 25 and 51 μg/L treatments at

substantial levels. Obviously, intersex and sex reversal are simply different points on a continuum ofpotential effects of an EDC on the gonad, and intersex tends to be more prevalent in lower concentrati ons,

which might lead to greater statistical power. However, intersex testes are routinely found in control

medaka, thus lowering statistical power and potentially confounding data interpretation.

Subadult Pathology (Tables 8

– 11) :

The patholo gies that appeared in F1 XY medaka (Table 8 ) that were related to exposure to 4tOP

included asynchronous development of the testes, hypoplasia of the gonad, increased severity and/or

prevalence of testicular oocytes, mononuclear cell infiltrates in the te stes, and basophilia of the liver.

Asynchronous development of the testis was characterized by the presence of relatively immature

spermatocysts (e.g., those containing primary or secondary spermatogonia) in areas of the testis where

they are not normally conspicuous (i.e., adjacent to the central duct). In essentially all affected testes, the

anatomic arrangement of the efferent duct system was irregularly formed to varying degrees. These

occurred at the LOEC of 13 μg/L with a severity of 1 and increase d to a severity score of 3 at 51 μg/L.

Testicular hypoplasia presented as an excessively small testis as compared to the testes of control males.

In some testes, this diagnosis was accompanied by asynchronous development. Hypoplasia was detected

with a severity score of 1 at 25 μg/L and increased to a score of 2 upon exposure to 51 μg/L 4tOP.

Several of these findings would support 4tOP acting on the ER . The increased testicular oocytes and

hypo plasia are indicative of an estrogenic EDC’s effects on the testes. In addition, it has been speculated

that the basophilia seen in the liver upon exposure to an estrogen is the result of vitellogenin synthesis in

the liver induced by the estrogen, in thi s case 4tOP.

In F1 XX fish (Table 9 ), several pathologies were determined to be statistically significant,

however almost all were of minimal severity and not expected to have negative impacts on the individual.

These findings included proteinaceous flu id in the ovary and kidney, nephropathy, kidney tubule

dilatation, and basophilia of the liver. All were seen at the LOEC of 51 μg/L 4tOP except basophilia in

the liver which had a LOEC of 102 μg/L and was the only finding with a severity score of 2.

The pathologies that appeared in F2 XY medaka (Table 10 ) that were related to exposure to 4tOP

included asynchronous development of the gonad, increased spermatogonia, interstitial fibrosis in the

gonad, testicular oocytes, proteinaceous fluid in the gona d, and eosonophilia of the tubular epithelium of

the kidney. The pathologies observed in the gonad of XY medaka seemed to be most often related to an

estrogenic EDC and its alterations of the phenotype of the gonad to a feminized state

.

In F2 XX medaka (Table 11 ), only two pathologies were statistically significant: proteinaceous

fluid in the kidney and basophilia of the liver. The biological significance of these is not entirely

understood but doesn’t seem to be characteristic of an estrogenic MOA. O ddly enough, basophilia of the

liver was seen in both F1 and F2 XX fish (and may have increased in severity in F2), but was not seen in

F2 XY medaka even though basophilia was observed in the F1 XY fish. If it is indicative of vitellogenin

synthesis in th e liver, it would have been expected to be observed in both F1 and F2 XY medaka

considering vitellogenin mRNA was indeed induced in the males in both generations at the concentrations

of 4tOP tested.

Subdult Weight (Figure 10) :

The only effects on weigh t seen with exposure to 4tOP in either F1 or F2 generations were

potential increases in F1, XY fish in the 6, 23, and 51 μg/L treatment groups and XX fish in the 13 μg/L

treatment. These were all statistically significant, however, they both may have been spurious, the result

of lower than normal weight in the controls, particularly the XY controls, which had a mean of 76 mg

compared to a typical mean of approximately 100 mg as found in the F2, XY controls. Taking the

statistical significance at face valu e, it is unclear how to interpret an increase in weight in either XX orXY medaka at concentrations of 4tOP that are lower than the typical LOECs of the other endpoints

measured in the 4tOP MMT. Therefore, these effects are not considered adverse outcomes resulting from

4tOP exposure.

Subadult Length (Figure 11) :

There were virtually no effects across treatments, genotypic sex, or generation on the length of

the animals. There was one isolated treatment group (F1, XY fish, 13 μg/L) that was statistical ly different

than controls, but it was in fact slightly longer. Considering there was no observed trend in the higher

concentrations, this seems most likely to be spurious.

Subadult Survival (Table 12

- 13) :

4tOP did not have an effect on survival at the concentrations tested from 4 wpf through 8 wpf

(Table 13 ). However, 4tOP did lower the survival at the early life stages (hatch through 4 wpf ; Table 12 )

at the highest concentration, 102 μg/L, in the F1 generation. This treatment group also had low fecundity

in F1. The combination of low fecundity and low early life stage survival prohibited this treatment (102

μg/L) from continuing into the F2 generation.

Subadult Hatch (Table 14) :

The only effect seen in the 4tOP MMT on hatch was in the 102 μg/L treatment of the F1

generation. This treatment had a hatch of 39% as compared to 85% in the controls of F1 generation. This

treatment group also had low fecundity and low early life stage survival in F1. The combination of low

fecundity, followed b y low hatch success, and then ending with low early life stage survival prohibited

this treatment (102 μg/L) from continuing into the F2 generation.Table 1. Summary table of LOECs for the various biological data collected duri ng the MED 4tOP MMT.

LOECs are color coded with more intense colors for lower concentrations. The direction of the change from controls is indica ted by the direction of the arrow

and statistical test used is abbreviated in the cell as well. JT=Jonckheere

- Terpstra, D=Dunnett, Du=Dunn 4tOP MMT LOEC Summary 4tOP (ug/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

subadult (7 wpf) subadult (7 wpf) adult (15 wpf) adult (15 wpf) adult (15 wpf) 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 6 0.6

Weight LOEC>102 LOEC> 102 LOEC>51 LOEC>51 LOEC>102 JT: 102 ↑ JT: 51 ↑ LOEC>102 LOEC>51 LOEC>51 3 13 1.5

Length LOEC>102 LOEC>102 LOEC>51 LOEC>51 LOEC>102 JT:102 ↑ J T: 13 ↑ LOEC>102 LOEC>51 LOEC>51 4 25 2.6

Survival LOEC>102 LOEC>51 LOEC>102 LOEC>102 LOEC>102 LOEC>102 LOEC>51 LOEC>51 5 51 5.6

Hatch 102 ↓ LOEC>51 6 102 8.5

Papillae D,JT: 25 ↓ LOEC>102 Du, JT: 13 ↓ LOEC>51 LOEC>102 LOEC>102 D,JT:102 ↓ LOEC>102 JT:13 ↓ LOEC>51

Vtg Du, JT: 25 ↑ Du, JT: 51 ↑ Du, JT: 25 ↑ JT : 51 ↑ D,JT: 51 ↑ LOEC>102 D, JT: 51 ↑ LOEC>102 51 ↑ LOEC>51

Gonad

Phenotype 25 ↑ LOEC>102 25 ↑ LOEC>51 LOEC>102 LOEC>102 13 ↑ LOEC>102 51 ↑ LOEC>51

Fecundity LOEC>102; trend ↓ 102 ↓ 51↓; not by wk

Fertility LOEC>102; trend ↓ 102 ↓ 51↓; esp wk11

Highest

Conc 102 51 102 102 51Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation,

and the whisker is the standard error of the number of eggs spawned per

day by each replicate. 4tOP F0 Reproduction MED MMT

0 6 13 25 51 102

Concentration (ug/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Raw Data 4tOP F1 Reproduction MED MMT

0 6 13 25 51 102

Concentration (ug/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Raw Data



### *

* 4tOP F2 Reproduction MED MMT

0 6 13 25 51

Concentration (ug/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Raw Data



### *

*Figure 2, adult vitellogenin. Gender specific vitellogenin gene expression of

adult medaka in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation

and the whisker is the standard error of the vitellogenin expression.

Vitellogenin gene expression is graphed re lative to the gender specific

controls. Significance is indicated by a letter: JT = Jonckheere

- Terpstra and

D = Dunnett’s. 4tOP F1 Adult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160 180

XY

XX

D, JT

D, JT 4tOP F0 Adult Vitellogenin MED MMT

Concentration (mg/L)Vtg mRNA Relative to Controls

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160 180

XY

XX

D

JT JT

D 4tOP F2 Adult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 6 13 25 51 102 0 50 100 150 200 250 300

XY

XX

Du, JTno viable F2 fishFigure 3, adult secondary sex characters. Gender specific secondary sex

characters (SSC) of adult (15 wpf) medaka i n F1 (upper left), F2 (upper

right), and F0 (lower left) generations after exposure to 4tOP. The central

line is the mean, the box is the standard deviation and the whisker is the

standard error of the number of anal fin papillae, that is the SSC. D or J T =

significantly different from controls as determined by Dunnett’s test or

Jonckheere

- Terpstra trend test, respectively. 4tOP F1 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160

XY

XX

D,JT

4tOP F0 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160

XY

XX 4tOP F2 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160

XY

XX

D,JT D,JT JTno viable F2 fishFigure 4, adult gonad phenotype. Gonad phenotype of XY adult (15 wpf)

medaka in F1 (upper left), F2 (upper right), and F0 (lower left) generations

after exposure to 4tOP. The stacked bar graphs show the percent of

individuals with testis, intersex, or ovary. The number in the boxes is the

number of individuals in each treatment with that gonad phenotype that is

the n. Intersex gonad is defined as a single gonad with any amount of both

ovarian and testicular tissue with no measurement of relative proportion.

Sex reversal is the presence of only ovarian tissue in a XY fish. 4tOP F1 Adult XY Gonad Phenotype MED MMT

Concentration (ug/L) . 0 6 13 25 51 102

. 0 20 40 60 80 100

6 6 3 4

4 4

4 2 2

1 intersex

tesits

ovary Intersex Sex Reversal

Concentration RSCABS p RSCABS p

0 na na

6 na na

13 0.04 na

25 0.01 na

51 0.0001 na

102 <0.00001 0.001

4tOP F0 Adult XY Gonad Phenotype MED MMT

Concentration (ug/L) . 0 6 13 25 51 102

. 0 20 40 60 80 100

6 6 1 1 1 1

5 5 5 5

intersex

tesits

ovary 4tOP F2 Adult XY Gonad Phenotype MED MMT

Concentration (ug/L) . 0 6 13 25 51 102 0 20 40 60 80 100

6 5

5 5 2 1 1

1 intersex

tesits

ovary Intersex Sex Reversal

Concentration RSCABS p RSCABS p

0 na na

6 na na

13 na na

25 0.1 na

51 0.001 0.07

102 na na

### Table 2. Significant pathologies as indicated by RSCABS in adult XY medaka of the 4tOP MED MMT

F0 XY F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc SeverityGONAD Interstitial Fibrosis 102 μ g/L 1 102 μ g/L 1

- - - - - - - -

Proteinaceous Fluid 102 μ g/L 1 102 μ g/L 1

- - - - - - - - Stage

- - - - 51 μ g/L 3 102 μ g/L 4 51 μg/L 2 51 μg/L 2

Testicular Oocytes

- - - - 13 μ g/L 1 102 μ g/L 4 51 μg/L 2 51 μg/L 2

Asynchronous Development

- - - - - - - - 25 μg/L 1 51 μg/L 3

Cell Infiltrate, Mononuclear

- - - - - - - - 51 μg/L 1 51 μg/L 1

Increased Spermatagonia

- - - - - - - - 25 μg/L 1 25 μg/L 1

Spermatogonia, Vitellogenin

- - - - - - - - 13 μg/L 1 51 μg/L 2KIDNEY Nephropathy 102 μg/L 2 102 μg/L 2 102 μ g/L 1 102 μ g/L 1 51 μ g/L 3 51 μ g/L 3

Proteinaceous Fluid 102 μg/L 2 102 μg/L 2 102 μ g/L 1 102 μ g/L 1

- - - -

Tubule Epithelium Eosinophilia

- - - - 102 μ g/L 3 102 μ g/L 3

- - - - Glomerulomegaly

- - - - - - - - 51 μ g/L 3 51 μ g/L 3

Hematopoietic Hyperplasia

- - - - - - - - 13 μ g/L 1 13 μ g/L 1

Mineralization, Tubules

- - - - - - - - 6 ug/L 1 1 6 ug/L

Li ver Basophilia 102 μg/L 2 102 μg/L 2 51 μ g/L 2 102 μ g/L 3

- - - -

S wim Bladder Hyperplasia 102 μg/L 1 102 μg/L 1

- - - - - - - -



### Table 3. Significant pathologies as indicated by RSCABS in adult XX medaka of the 4tOP MED MMT

F0 XY F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

Gonad Asynchronous Develop

- - - - - - - - 51 μ g/L 2 51 μ g/L 2

Kidney Proteinaceous Fluid 102 μg/L 1 102 μg/L 1

- - - - - - - -

Li ver Cystic Degeneration 102 μg/L 1 102 μg/L 1

- - - - - - - -Figure 5, adult weight. Gender specific weights of adult (15 wpf) medaka in

F1 (upper left), F2 (upper right), and F0 (lower left) generations after

exposed to 4tOP. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the weig ht measured in

mg. JT = significantly different from controls as determined by Jonckheere

Terpstra trend test. 4tOP F1 Adult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 6 13 25 51 102 0 100 200 300 400 500 600 700 800 900

XY

XX

JT JT

4tOP F0 Adult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 6 13 25 51 102 0 100 200 300 400 500 600 700 800 900

XY

XX

JT 4tOP F2 Adult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 6 13 25 51 102 0 100 200 300 400 500 600 700 800 900

XY

XXFigure 6, adult weight. Gender specific lengths of adult (15 wpf) medaka in

F1 (upper left), F2 (upper right), and F0 (lowe r left) generations after

exposed to 4tOP. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the weight measured in

mg. JT = significantly different from controls as determined by Jonckheere

Terpst ra trend test. 4tOP F1 Adult Length MED MMT

Concentration (ug/L)Length (mm)

0 6 13 25 51 102 0 5 10 15 20 25 30 35 40

JT

XX

XY JT JT JT

4tOP F0 Adult Length MED MMT

Concentration (ug/L)Length (mm)

0 6 13 25 51 102 0 5 10 15 20 25 30 35 40

XX

XY JT 4tOP F2 Adult Length MED MMT

Treatment (ug/L)Length (mm)

0 6 13 25 51 0 5 10 15 20 25 30 35 40

XX

XYTable 4. Survival from 8 wpf to 15 wpf.

Adult Survival (mean %, (N))

Treatment

Generation control 6μg/L 13μg/L 25μg/L 51μg/L 102μg/L

F0 100 (6) 100 (6) 100 (6) 100 (6) 92 (6) 100 (6)

F1 100 (6) 100 (6) 100 (6) 100 (6) 92 (6) 92 (6)

F2 92 (6) 92 (6) 83 (6) 92 (6) 75 (6)Figure 7, subadult vitellogenin. Gender specific vitellogenin gene

expression of subadult medaka in F1 (left), and F2 (right) generations. The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the vitellogenin expression. Vitellogenin gene

expression is graphed relative to the gender specific controls. S ignificance is

indicated by a letter: JT = Jonckheere

- Terpstra, Tam = Tamhane and D =

Dunnett’s. 4tOP F1 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 6 13 25 51 102 0 100 200 300 400 500

XY

XX

D, JT D, JT D, JT

D, JT

D, JT 4tOP F2 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 6 13 25 51 102 0 100 200 300 400 500

XY

XX

JT, Tam D, JT

D, JTFigure 8, subadult secondary sex characters. Gender specific secondary

sex characters (SSC) of sub adult (7 wpf) medaka in F1 (left), and F2 (right )

generations after exposure to 4tOP . The central line is the mean, the box is

the standard deviation and the whisker is the standard error of the number of

anal fin papillae that is the SSC. D, Du or JT = significantly different from

controls as determined by a Dunnett test, Dunn’s test or Jonckheere

- Terpstra

trend test, respectively. 4tOP F1 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 6 13 25 51 102 0 10 20 30 40 50 60 70 80 90

XY

XX

D,JT D,JT

D,JT 4tOP F2 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 6 13 25 51 102 0 10 20 30 40 50 60 70 80 90

XY

XXno viable F2 fish

Du, JT Du, JT Du, JTFigure 9, subadult gonad phenotype. Gonad phenotype of XY subadult

medaka in F1 (left) and F2 (right) after exposure to 4tOP . The stacked bar

graphs show the percent of individuals with testis, intersex, or ovary. The

number in the boxes is the number of individuals in each treatment with that

gonad phenoty pe that is the n. Intersex gonad is defined as a single gonad

with any amount of both ovarian and testicular tissue with no measurement

of relative proportion. 4tOP F1 Subdult XY Gonad Phenotype MED MMT

Concentration (ug/L) . 0 6 13 25 51 102

. 0 20 40 60 80 100

2

19 19 16 13

11 4

7 5 1

1

1 5

intersex

tesits

ovary 4tOP F2 Subadult XY Gonad Phenotype MED MMT

Concentration (ug/L) . 0 6 13 25 51 102 0 20 40 60 80 100

4

23 21 6

15

15 10 9 3

3 3

intersex

tesits

ovary

### Table 5. Significant pathologies as indicated by RSCABS in subadult XY medaka of the 4tOP MED MMT

F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc SeverityGONAD Asynchronous Development 13 μ g/L 1 51 μ g/L 3 13 μ g/L 2 13 μ g/L 2

Hypoplasia 25 μ g/L 1 51 μ g/L 2

- - - - Testicular Oocytes 25 μ g/L 2 51 μ g/L 4 13 μ g/L 2 25 μ g/L 4

Cell Infiltrate, Mononuclear 51 μ g/L 1 51 μ g/L 1

- - - -

Increased Spermatagonia

- - - - 51 μ g/L 2 51 μ g/L 2

In terstitial Fibrosis

- - - - 25 μ g/L 1 51 μ g/L 3

Stage

- - - - 25 μ g/L 2 25 μ g/L 2

Pr oteinaceous Fluid

- - - - 51 μ g/L 1 51 μ g/L 1

KIDNEY Tub Epith Eosinophilia

- - - - 51 μ g/L 3 51 μ g/L 3

Li ver Basophilia 102 μ g/L 2 102 μ g/L 2

- - - -



### Table 6. Significant pathologies as indicated by RSCABS in subadult XX medaka of the 4tOP MED MMT

F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Proteinaceous Fluid 51 μ g/L 1 51 μ g/L 1

- - - -

KIDNEY Nephropathy 51 μ g/L 1 51 μ g/L 1

- - - -

Proteinaceous Fluid 51 μ g/L 1 51 μ g/L 1 51 μ g/L 2 51 μ g/L 2

Tubule Dilatation 13 μ g/L 1 13 μ g/L 1

- - - -

Li ver Basophilia 102 μ g/L 2 102 μ g/L 2 51 μ g/L 3 51 μ g/L 3Figure 10, subadult weight. Gender specific weight of sub adult (7 wpf)

medaka in F1 (left), and F2 (right ) generations after exposure to 4tOP

. The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the weight

. D = significantly different from controls as

determined by a Dunnett test test

. 4tOP F1 Subadult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160 180 200 220 240 XY

XX

D

D D

D 4tOP F2 Subadult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 6 13 25 51 102 0 20 40 60 80 100 120 140 160 180 200 220 240

XY

XXno viable F2 fishFigure 11, subadult length. Gender specific length of sub adult (7 wpf)

medaka in F1 (left), and F2 (right ) generations after exposure to 4tOP . The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the le ngth (mm)

. 4tOP F1 Subadult Length MED MMT

Concentration (ug/L)Length

0 6 13 25 51 102 0 5 10 15 20 25 30 4tOP F2 Subadult Length MED MMT

Concentration (ug/L)Length

0 6 13 25 51 0 5 10 15 20 25 30

XY

XXTable 12. Early life survival (hatch to 4 wpf)

Survival (mean % )

Treatment

Generation control 6μg/L 13μg/L 25μg/L 51μg/L 102μg/L

F1 85 76 88 88 78 41

F2 87 87 82 88 91

Table 13. Subadult survival (4 wpf to 8 wpf)

Survival (mean % )

Treatment

Generation control 6μg/L 13μg/L 25μg/L 51μg/L 102μg/L

F1 97 97 98 97 97 87

F2 93 98 95 97 90

Table 14. Hatch

Hatch (mean % )

Treatment

Generation control 6μg/L 13μg/L 25μg/L 51μg/L 102μg/L

F1 85 82 88 86 78 39

F2 87 88 82 88 90Test Chemical: 4

- tert

- octylphenol (4tOP)

MOA: estrogen (estrogen receptor agonist)

Protocol: full research MMT

Lab: NIES (Japan)

Summary of Test Chemical and MOA:

4tOP is an active degradation product of octylphenol ethoxylates , which are widely used

nonionic surfactants used in the manufacture of cleaning agents, plastics, paper, cosmetics and food

products. 4tOP has been shown to be estrogeni c in both in vitro systems and in fish exposures including

medaka. For instance in a 60 day exposure starting with fertilized medaka eggs, 4tOP induced hepatic

vitellogenin production and altered sexual differentiation (intersex gonads) at a LOEC of 11.4 μ g/L.

Needless to say, the estrogenic properties of 4tOP are well documented as well as its presence in many

aquatic environments.

Summary of Protocol:

The MMT completed by NIES t esting the effects of 4tOP was done with the MMT protocol that

has been detailed previously (pp. ). Briefly, the 4tOP MMT protocol spanned 2 1⁄2 generations, in which

in the F0 generation chemical exposure was initiated with spawning pairs as a chemical loading phase

only, that is, no data was collected from F0 fish . In the F1 and F2 generations, fish were continuously

exposed from embryo through adult spawning. Fish were sampled at two major life

- stages; subadults (in

the 8 th wpf) in the F1 and F2 generations, and mature adults (at the start of the 15th wpf) inF1 and F2.

Rep roduction was evaluated over 3 weeks from 13 wpf to 15 wpf.

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria) . The

proportionate chemical concentration diffe rence between treatments was 0.5. Reproduction in the two

generatio ns ( F1, and F2) consisted, nominally, of 6 breeding pairs per concentration and 6 breeding pairs

for controls. These breeding pairs were al so the fish sampled as adults. At the subadult (8 wpf ) sampling,

8 of the 10 fish per aquaria were randomly sacrificed to a) produce the subadult samples and to b) leave

behind the breeding pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair in

F1 and F2; in adults in F1 and F2 , VTG liver concentration and SSC ; and in subadults i n F1 and F2, VTG

liver concentration, SSC, weight

, and length were record ed. In addition, hatch was recorded.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with the direction of the effect indicated. The

LOEC is color

- coded with more intense colors for lower concentrations of 4tOP

. There were no viable

fish available in the 100 μ g/L treatment of F2, so only effects at 50 μ g/L or lower were observable. In

addition, the LOECs obtained at the subadult samplings were lower than those of the adult samplings.

However, whether this was a result of a g reater sensitivity at this lifestage or a result of the statistical

impact of greater sample size per replicate at the subadult sampling is difficult to determine from the data.

The LOEC for fecundity and fertility, arguably the most important endpoints w ere 100 μ g/L in F1. The

LOECs across the entire 4tOP MMT ranged from 6.25 to 100 μ g/L, seemingly centered around 50 μ g/L,

and predominately affected XY fish, as expected with an estrogenic EDC.The suite of biological impacts on the fish within the MMT could be linked across levels of

biological organization to the well documented mode of action of 4tOP as an ER agonist. For instance at

the population/organismal level, fecundity, was reduced starting at 100 μ g/L in F1. At the

individual/tissue level of organization, feminized testes were observed in XY individuals exposed to 100

μ g/L 4tOP. At the same time and further following an estrogenic mode of action now at the molecular

level, 4tOP induced vitellogenin in XY fish as low as 6.25 μ g/L. So again a ll the observed effects

“passed muster” as being attributable to 4tOP binding to and activating the estrogen receptor.

Reproduction (Tables 2

- 3) :

In F1, the 100 μg/L treatment had lower fecundity and fertility than controls. In addition to being

statistically significant, the magnitude of the decrease (29 eggs per pair

- day versus 6 eggs per pair

- day)

was substantial and resulted in not enough embryos to continue this treatment group into F2. In a ddition,

the pairs exposed to 50 μg/L, while not statistically dif ferent from controls in F1, had lower fertility than

what would be anticipated in normal reproduction.

In F2, there were no 100 μg/L exposed fish available for breeding pairs. There were no effects to

report, except an extremely high fertility percentage in the 6.25 μg/L which is of unclear importance.

Adult Secondary Sex Character s (Figure 1) :

In F1, 3 of 6 XY fish in the 100 μg/L exposed trea tment did not have any papillae (which is the

female phenot ype) while the XY fish in the 50 μg/L group also had fewer papillae than controls. In

addition, F2, XY fish in the 50 μg/L treatment group had reduced numbers of anal fin papillae as

compared to c ontrols (means of 120 versus 85) at similar values as seen in F1.

Subadult Vitellogenin (Tabes 4

- 5) :

In subadult fish, 4tOP caused significant increases in vitellogenin in the liver of XY fish in the

12.5 μg/L through 100 μg/L treatment groups of the F1 generation. In the 100 μg/L treatment, the

vitellogenin concentration in the liver increased 87 times over that of controls while more modest

increases were observed in the 12.5 (4 times), 25 (6 times), and 50 (9 times) μg/L treatments. In addition

to the effects on XY medaka, 4tOP expos ure also increased vitellogenin in XX individuals in the 6.25

through 100 μg/L treatments by a factor of 1.5

– 3.3 times.

A similar trend is also evident in the subadults of the F2 generation. The LOEC in the XY fi sh

was 6.25 μg/L 4tOP where th e level of induction was about 4 times that of controls while in the highe st

treatment (50 μg/L), the level of induction was about 17 times that of controls. Like the F1 generation,

there was a n increase in vitellogenin starting at 6.25 μg/L in XX fish in the F2s.

Subadult Secondary Sex Characters (Figure 2) :

In F1 XY fish sampled as subadults, the anal fin papillae were reduced in all the concentrations of

4tOP tested (6.25

- 100 μg/L ) by a considerable amount. The mean in for controls was 71 while for 6.25,

12.5, 25, 50, and 100 μg/L respectively, the means were 10, 23, 15, 12, and 1. Interestingly enough, this

apparently strong affect on papillae was not as evident in the F2, XY fish sampled as subadults. While

the controls mean was essentially the same (69 papillae), the means for each treatment group was much

higher than observed in the same treatment of F1. In fact, only 25 and 50 μg/L 4tOP treatments were

statistically lower than controls, but these treatme nts still had almost normal mean values: 53 and 50,

respectively.Subadult Gonad Phenotype (Tables 6

- 7) :

In F1, it appears that NIES observed changes in the gonad phenotype of XY fish at 100 μg/L . Of

the 26 XY fish sampled, 22 were reported to have “immature” gonads. At this time, it is unclear whether

the gonads were sectioned and assessed histologically or if only gross observations were made. Therefore

it is also unclear what the diagnosis of “immature” truly represents. Based upon the data pro vided by

NIES, it seems safe to conclude that at least at a concentration of 4tOP of 100 μg/L , the gonad phenotype

was altered, presumably feminized.

Because of very low reproduction in the F1, 100 μg/L treatment group, the highest concentration

presen t in F2 was 50 μg/L . At this concentration (or lower), there was data to suggest effects of 4tOP on

the gonad phenotype.

Subdult Weight (Figure 3) :

In F1, 4tOP exposure did not affect weight at any of the concentrations tested. In F2, the LOEC

was 25 μg/L with a slight increase in weight from a mean of 199 mg in controls to 233 mg in the 25 μg/L

treatment and 224 mg in the 50 μg/L treatment. The data submitted by NIES did not categorize the

individual weight measurements as coming from a XX or XY fish . Therefore, it is not possible to

determine whether the increase was specific to one genotype or not.

Subadult Length (Figure 4) :

In F1, 4tOP exposure did not affect length at any of the concentrations tested. In F2, the lengths

were increased slightly at exposures of 6.25 μg/L and above. The control mean length in F2 was 26.7 mm

as compared to 27.4, 27.3, 27.6, and 27.3 in the 6.2 5 μg/L , 12.5 μg/L , 25 μg/L , and 50 μg/L treatments,

respectively. As a note, the mean control length in F2 was also shorter than that observed in the control

group of F1, seeming to suggest that the LOEC in F2 was influenced by shorter than normal control fish

in this generation. Like weight, the data submitted by NIES did not categorize the individual length

measurements as coming from a XX or XY fish. Therefore, it is not possible to determine whether the

increase was specific to one genotype or not.

Hatch (Table 8) :

Exposure to 4t OP did not reduce hatch at any of the concentrations tested in either the F1 or F2

generation. In F1, the mean percentage hatch in the control groups was 92.2%, and a high hatch

percentage (89%) was maintained in the highest treatment (100 μg/L ). F2 was similar with no

statistically significant decreases in hatch observed. The control hatch was at 87% and the high dose, 50

μg/L in F2, had a hatch of 77%.Table 1. Summary table of LOECs for the various biological data collected during the NIES 4tOP MMT.

4tOP MMT LOEC Summary 4tOP (ug/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

subadult (8 wpf) subadult (8 wpf) adult (15 wpf) adult (15 wpf) adult (15 wpf) 1 0 na

XY XX XY XX XY XX XY XX XY XX 2 6.25 na

Weight LOEC>100 25 ↑ 3 12.5 na

Length LOEC>100 6.25 ↑ 4 25 na

Survival LOEC>100 ? LOEC>50 ? LOEC>100 LOEC>100 LOEC>50 LOEC>50 5 50 na

Hatch LOEC>100 LOEC>50 6 100 na

Papillae 6.25 ↓ LOEC>100 25 ↓ LOEC>50 50 ↓ LOEC>100 50 ↓ LOEC>50

Vtg 25 ↑ 6.25 ↑ 6.25 ↑ 6.25 ↑

Gonad

Phenotype 100? LOEC>100 LOEC>50 LOEC>50

Fecundity 100 ↓ LOEC>50

Fertility 100 ↓ LOEC>50

Highest

Conc 100 50 100 100 50Table 1: Fecundity: number of eggs (average number per day) and fertility(%) in F1 generation

Treatment number of fertility

(μg/L) total eggs (%)

control 29 ± 4.9 99 ± 0.65

6.25 34 ± 4.3 96 ± 4.3

12.5 31 ± 6.4 98 ± 2.0

25 33 ± 3.9 98 ± 1.5

50 27 ± 8. 8 80 ± 40

100 6 ± 2. 0** 0.81 ± 0.79**

average±S. D.

Statistica lly significant differences from control group (*p<0.05, **p<0.01).

Only processed data exists

Table 2: Fecundity: number of eggs (average number per day) and fertility(%) in F2 generation

Treatment number of fertility

(μg/L) total eggs (%)

control 27 ± 3.3 96 ± 8.8

6.25 33 ± 5.8 99 ± 0.50*

12.5 25 ± 17 53 ± 50

25 32 ± 4.0 86 ± 32

50 32 ± 4. 3 96 ± 5.9

100

- -

average±S. D.

Statistica lly significant differences from control group (*p<0.05, **p<0.01).

Only processed data existsFigure 1, Adult secondary sex characters. Gender specific secon dary sex

characters (SSC) of adult (15 wpf) medaka in F1 (left), and F2 (right )

generations after exposure to 4tOP . The central line is the mean, the box is

the standard deviation and the whisker is the standard error of the number of

anal fin papillae that is the SSC. JT = significantly different from controls

as determined Jonckheere

- Terpstra trend test.Table 4: VTG concentration in liver at the 8 weeks post

- fertilization (F1 generation)

Treatment VTG(nglmg liver)

(μg/L) DMY(+) DMY(

- )

control 2.3 ± 2.8 490 ± 305

6.25 4.8 ± 4.9 720 ± 385*

12.5 9.4 ± 15* 840 ± 342*

25 13 ± 13** 1100 ± 354**

50 21 ± 20** 1300 ± 644**

100 200 ± 240** 1600 ± 1099**

average±S.D.

Statistically significant differences from control group (*p<0.05, **p<0.01).

Only processed data exists

Table 5: VTG concentration in liver at 8 the weeks post

- fertilization (F2 generation)

Treatment VTG(nglmg liver)

(μg/L) DMY(+) DMY(

- )

control 2 ± 1.7 470 ± 567

6.25 8 ± 8.0** 950 ± 282*

12.5 10 ± 7.9** 700 ± 515

25 19 ± 12** 1000 ± 366*

50 33 ± 27** 1300 ± 509*

100

- -

average±S.D.

Statistically significant differences from control group (*p<0.05, **p<0.01).

Only processed data existsFigure 2, subadult secondary sex characters. Gender specific secondary

sex characters (SSC) of sub adult (8 wpf) medaka in F1 (left), and F2 (right )

generations after exposure to 4tOP . The central line is the mean, the box is

the standard deviation and the whisker is the standard error of the number of

anal fin papillae that is the SSC. JT = significantly different from controls

as determined by a Jonckheere

- T erpstra trend test

.Table 6: Hist o pathology with XY

- male and XX

- female medaka at 8 wph (F1)

treatment

(μg/L) genetic sex N of

male

medaka gonad

testis immature ovary

control XY

- male 23 23

XX

- female 25 25

6.25 XY

- male 18 27

XX

- female 30 20

12.5 XY

- male 18 16 2

XX

- female 30 30

25 XY

- male 25 21

XX

- female 22 25

50 XY

- male 27 22 5

XX

- female 19 19

100 XY

- male 26 4 22

XX

- female 22 22

Statistically significant differences from control group (** p <0.01, * p <0.05).

Table 7: Hist o pathology with XY

- male and XX

- female medaka a t 8 wpf (F2)

treatment (μg/L) genetic sex N of male

medaka gonad

testis immature ovary

control XY

- male 27 27

XX

- female 21 21

6.25 XY

- male 26 26

XX

- female 22 22

12.5 XY

- male 25 25

XX

- female 23 23

25 XY

- male 17 16 1

XX

- female 31 31

50 XY

- male 28 27 1

XX

- female 20 20

100 XY

- male 0

XX

- female 0

Statistically significant differences from control group (** p <0.01, * p <0.05).Figure 3, subadult weight. Weight of sub adult (8 wpf) medaka in F1 (left),

and F2 (right ) generations after exposure to 4tOP . The central line is the

mean, the box is the standard deviation and the whisker is the standard error

of the weight

. JT = significantly different from controls as determined by a

Jonckhee re

- T erpstra trend test

.Figure 4, subadult length. Length of sub adult (8 wpf) medaka in F1 (left),

and F2 (right ) generations after exposure to 4tOP

. The central line is the

mean, the box is the standard deviation and the whisker is the standard error

of the length (mm)

. JT = significantly different from controls as determined

by a Jonckheere

- T erpstra trend test

.Table 8. Hatch

Hatch (mean % )

Treatment

Generation control 6 .25 μg/L 12.5 μg/L 25 μg/L 50 μg/L 100 μg/L

F1 92 89 91 86 88 89

F2 87 89 76 70 77 NATest Chemical: 1,1,1

- trichloro

- 2

- (2

- chlorophenyl)

- 2

- (4

- chlorophenyl)ethane ; o,p’

- DDT

MOA: estrogen (estrogen receptor agonist)

Protocol: full research MMT

Lab: CERI (Japan)

Summary of Test Chemical and MOA:

O, p’

- DDT is a persistent pesticide that is known to bind to the ER and induce estrogenic

responses in various biological systems including the medaka.

Summary of Protocol:

The MMT completed by CERI t esting the effects of o,p’

- DDT was done with a full research

MMT protocol

. Briefly, the o,p’

- DDT MMT protocol spanned 2 1⁄2 generations, in which in the F0

generation chemical exposure was initiated with adult spawning pairs and continues through the end of

the reproductive assessment . In the F1 and F 2 generations, fish were continuously exposed from embryo

through adult spawning. Fish were sampled at two major life

- stages; subadults (approximately 8 wpf) in

the F1 and F2 generations, and mature adults ( approximately 16 wpf) in F0, F1, and F2. Reprod uction

was evaluated for 5 consecutive days per week over 4 weeks starting at 10 wpf

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 repli cate units (i.e. aquaria). The

proportionate chemical concentration diffe rence between treatments was approximately 0.3. The average

measured E2 concentrations in the test were 0.03, 0.07, 0.22, 0.69, and 1.9 μ g/L . Reproduction in all

three generations (F 0, F1, and F2) consisted, nominally, of 6 breeding pairs per concentration and 6

breeding pairs for controls. These breeding pairs were also the fish sampled as adults.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with bot h the direction of the effect, and the

statistical test used indicated. The LOEC is color

- coded with more intense colors for lower

concentrations of o,p’

- DDT

. The most sensitive endpoint, at least in the context of the endpoint with the

lowest LOEC, was hepatic vitellogenin production in XX fish which had a LOEC of 0.07 μ g/L o,p’

- DDT.

The magnitude of the increased vitellogenin was substantial ( at the LOEC of 0.07 μ g/L was double that of

controls and at 1.9 μ g/L the concentration of vitellogenin was appr oximately 7 times that of controls ),

however the biological significance of increased vitellogenin in female fish is difficult to describe. The

primary endpoint for the MMT, reproduction, was impacted by exposure with lower fecundity in F2, with

a LOEC of 1.9 μ g/L, and lower fertility in both F1 and F2, again with a LOEC of 1.9 μ g/L. The AOP

sensitive endpoints of SSC (anal fin papillae) and vitellogenin production were altered from XY control

fish by exposure at 1.9 μ g/L o,p’

- DDT as would be predicted wi th an ER agonist. In addition, there were

feminized testes present in exposed fish with a LOEC of either 0.69 or 1.9 μ g/L.

Reproduction (Figure 1) :

No changes in reproduction, either in fecundity or fertility, were observed in the F0 generation.

There was decreased fertility in the 1.9 μ g/L treatment in F1 while this same treatment did not have

statistically lower fecundity than controls. The lower mean fertility in the 1.9 μ g/L treatment was mostly

a result of two pairs of fish producing non

- fertile eggs including pair that had a mean fecundity of 19.6

eggs per day but none were fertile. In F2, both fecundity and fertility were lower in the 1.9 μ g /L

exposure than in control pairs. While the control fecundity and fertility were 20.3 and 17.6, exposure to1.9 μ g/L o,p’

- DDT reduced fecundity and fertility to 14.0 and 6.9 eggs per day, respectively. Therefore

the reproductive LOEC was 1.9 μ g/L and th ere was at least some indication that fertility might have been

reduced to a greater magnitude than fecundity.

Adult Vitellogenin (Figure 2)

In F0 adult fish, o,p’

- DDT exposure of 1.9 μ g/L increased the amount of hepatic vitellogenin in

XY individual s, but had no effect on the other XY fish in the lower treatments. In addition, XX fish of F0

were unaffected by exposure at any of the concentrations tested in the MMT.

In F1 XY fish, the LOEC was 0.69 μ g/L with a modest increase of vitellogenin to 2 6 ng/mg liver

tissue as compared to controls which had a mean less than 1 ng/mg liver and most were below the

detection limit of the vitellogenin assay. On the other hand, the XY fish of the 1.9 μ g/L treatment had a

mean of 5260 ng/mg liver which was even well above that of control XX fish. The LOEC for increased

vitellogenin in XX fish was 0.07 μ g/L o,p’

- DDT. However, this is a bit misleading in that the magnitude

of the increase was very modest and the LOEC calculation was “driven” in part by a relativ ely high mean

of 1499 ng/mg in the 0.07 μ g/L treatment that did not continue into the next higher treatment (0.22 μ g/L

and 0.69 μ g/L) which had mean vitellogenin concentrations (1229 and 11292 ng/mg liver) that were more

similar to the controls (1105 ng/mg liver). Therefore, the apparent sensitivity of this endpoint in F1XX

adults was somewhat skewed by a relatively high mean in an intermediate treatment in combination with

the step

- down nature of the Jonckheere

- Terpstra trend test

.

In F2, XY fish had increased vitellogenin starting at 0.22 μ g/L o,p’

- DDT which had a modest

mean of 5 ng/mg liver tissue while the 0.69 and 1.9 μ g/L treatments had means of 300 and 6973 ng/mg

liver, respectively. These mean hepatic vitellogenin values were compared to the mean of XY controls of

0.5 ng/mg. Also, the LOEC for XX fish in F2 was 0.69 μ g/L which had slightly elevated vitellogenin

(1150 ng/mg liver) over that observed in controls (955 ng/mg). In addition, the mean of the 1.9 μ g/L

treatment was further elevated to 2032 ng/mg liver.

Adult Secondary Sex Character s (no figure) :

There were no effects observed on SSC in any of the generations or treatments.

Adult Gonad phenotype , XY fish only (Figure 3) :

No intersex gonads were observed in F0 fish. On the other hand, all of the adult XY fish (5 total)

in the 1.9 μ g/L treatment had intersex testes but none of the 0.69 μ g/L treatment (the next lowest) had

intersex gonads. Likewise n F2 adults, all of the XY medaka of the 1.9 μ g/L treatment (6 total) had

intersex gonads while 2 of 6 in the 0.69 μ g/L treatment had intersex gonads, making this treatment the

LOEC. In addition, there was 1 XY fish in the 0.22 μ g/L treatment that had an intersex testis.

Adult Pathology (no tables) :

No histopathology data was provided.

Adult Weight (no figure) :

No adult weight data was provided.

Adult Length (no figure) :No adult length data was provided.

Adult Survival (no figure) :

No adult survival data was pr ovided.

Subadult Vitellogenin (Figure 4) :

Like the adult life

- stage, the subadult XX fish had statistically significant induction of

vitellogenin in the liver. This affect was evident down to 0.07 μg/L o, p

- DDT, even if the magnitude of

the increased v itellogenin level was fairly small. At any rate, it seems unlikely that this would be a result

of or lead to an adverse outcome, like liver or kidney pathology, which is sometimes seen in males with

high levels of vitellogenin. Interestingly, the amount of vitellogenin per mg of liver tissue in the XX fish

exposed to the higher concentrations of o, p ’

- DDT was greater on a per mg basis than in the adult fish. It

is unclear why subadult XX fish would be induced into producing more vitellogenin then reprodu ctively

active adult XX fish by the same concentration of o, p

- DDT.

In both F1 and F2, XY subadults had a LOEC of 1.9 μg/L, which was higher than what was

observed in the adult fish in both F1 and F2. While the subadult LOECs were equal between the

generations, the magnitude of the effect was not; the XY fish exposed to 1.9 μg/L in F1 had a mean

vitellogenin level of 1554 ng/ mg of liver while the same treatment level in F2 had only 7 ng/mg of liver.

In addition, the group exposed to 0.69 μg/L in F1 had a mean vitellogenin level of 243 ng/mg of liver

which on the surface would appear to be a large increase over controls even t hough it was not statistically

significant. These findings in F1 should be viewed with caution however. Of the XY fish exposed to

0.69 μg/L in F1, 11 of the 12 had a mean of 0.5 ng/mg while a single individual had 2910 ng of

vitellogenin/mg of liver, whi ch by any statistical definition could be considered an outlier and obviously

increased the mean substantially. Similarly, in the F1 1.9 μg/L treatment, 9 of 12 XY fish were centered

around 5 ng/mg of liver while 3 of the 12 each had approximately 6000 ng of vitellogenin/mg of liver.

Again, by a statistical definition, these could be considered outliers, but since there are no explanations of

why they should be considered erroneous, they were included in the analysis.

Subadult Secondary Sex Characters (F igure 5) :

While there were no effects on SSC in the adults across the three generations of the MMT, there

was a decrease in anal fin papillae in both the F1 and F2 generation at the subadult life

- stage upon

exposure to 1.9 μg/L o, p ’

- DDT. In F1, the con trol mean for the XY fish was 51 while in the 1.9 μg/L

group the anal fin papillae per fish was reduced to 9; similarly the control in F2 was 52 while the 1.9 μg/L

treatment had a mean of 21. It appeared by comparing the adult and subadult data that 1) th e subadult

life

- stage is more sensitive than the adult life

- stage, which is probably a result of both biological

sensitivity and statistical “sensitivity” or more appropriately statistical power gained through the larger

‘n’ at the subadult sampling and 2) as the affected fish developed from subadults to adults they were able

to catch

- up (at least in the context of SSC) with their control brethren.

Subadult Gonad Phenotype (Figure 6) :

T he order of LOECs between F1 and F2 was the reverse at the subadult life

- stage of what it was

at the adult life

- stage. In F1, there was a XY fish that had an ovary and 3 of 11 that had intersex gonads

in the 0.69 μg/L treatment while an even stronger effe ct was seen in the 1.9 μg/L treatment, in which 3 of

12 XY fish had ovaries and 3 of 12 had intersex gonads. At the highest concentration, 1.9 μg/L, in F2,

there was 1 of 12 XY fish with an ovary and 10 of 12 with intersex gonads. The difference between thegenerations was subtle enough that it is difficult to conclude that there was an actual difference in

sensitivity between F1 and F2.

Suba dult Pathology (no tables) :

No histopathology data was provided.

Subdult Weight (Figure 7) :

There were no e ffects on the weight of subadults in any the treatment groups in either the F1 or

F2 generations.

Subadult Length (Figure 8) :

There was a very slight decrease in the 1.9 μg/L group in F2, but in none of the other treatment

levels in either the F1 or F2 generation.

Subadult Survival (Table 2) :

There was no effect from o, p ’

- DDT exposure on subadult survival in either the F1 or F2

generations.

Subadult Hatch (Table 3) :

There was no effect from o, p

- DDT exposure on embryo hatch in either the F1 or F2 generations.Table 1. Summary table of LOECs for the various biological data collected during the CERI o,p’

- DDT MMT.

LOECs for o, p

- DDT MMT DDT (ug/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

8 wph 8 wph 15 wph 15 wph 15 wph 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 0.032 0.004

Weight LOEC>1.9 LOEC>1.9 3 0.07 0.01

Length LOEC>1.9 1.9 ↓ 4 0.22 0.04

Survival LOEC>1.9 LOEC>1.9 5 0.69 0.17

Hatch LOEC>1.9 LOEC>1.9 6 1.9 0.43

Papillae 1.9 ↓ 1.9 ↓ LOEC>1.9 LOEC>1.9 LOEC>1.9

Vtg 1.9 ↑ 0.07↑ 1.9 ↑ 0.07↑ 1.9 ↑ LOEC>1.9 0.69 ↑ 0.07 ↑ 0.22 ↑ 0.69 ↑

Gonad Phenotype 0.69 ↑ LOEC>1.9 1.9 ↑ LOEC>1.9 LOEC>1.9 LOEC>1.9 1.9 ↑ LOEC>1.9 0.69 ↑ LOEC>1.9

Fecundity LOEC>1.9 LOEC>1.9 1.9 ↓

Fertility LOEC>1.9 1.9 ↓ 1.9 ↓

Highest Conc 1.9 1.9 1.9 1.9 1.9Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation,

and the whisker is the standard error of the number of eggs spawned per

day by each replicate. o,p'-DDT F0 Reproduction CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 5 10 15 20 25 30 35

Fecundity

Fertility o,p'-DDT F1 Reproduction CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 5 10 15 20 25 30 35

Fecundity

Fertility



# * o,p'-DDT F2 Reproduction CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 5 10 15 20 25 30 35

Fecundity

Fertility



# *

*Figure 2, adult vitellogenin. Gender specific vitellogenin gene expression of

adult medaka in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation

and the whisker is the standard error of the vitellogenin expression.

Significantly different from controls, as determined by the Jonckheere

Terpstra trend test, is indicated by *. o,p'-DDT F0 Adult Vitellogenin CERI MMT

Concentration (ug/L)Vitellogenin Protein (ng/mg liver)

0 0.032 0.07 0.22 0.69 1.9 0 2000 4000 6000 8000 10000 12000

XY

XX



## * o,p'-DDT F1 Adult Vitellogenin CERI MMT

Concentration (ug/L)Vitellogenin Protein (ng/mg liver)

0 0.032 0.07 0.22 0.69 1.9 0 2000 4000 6000 8000 10000 12000

XY

XX



# *

*

* * *

* o,p'-DDT F2 Adult Vitellogenin CERI MMT

Concentration (ug/L)Vitellogenin Protein (ng/mg liver)

0 0.032 0.07 0.22 0.69 1.9 0 2000 4000 6000 8000 10000 12000

XY

XX



## * *

* *

*Figure 3 , adult gonad phenotype. Gonad phenotype of XY adult medaka

in F1 (left) and F2 ( right) generations after exposure to o,p’

- DDT

. No data

from F0 is presented as all XY individuals had testis with no intersex. The

stacked bar graphs show the percent of individuals with testis, intersex, or

ovary. The number in the boxes is the number of individuals in each

treatment with that gon ad phenotype. Intersex gonad is defined as a single

gonad with any amount of both ovarian and testicular tissue with no

measurement of relative proportion.Figure 4, subadult vitellogenin. Gender specific hepatic vitellogenin in subadult medaka in F1 (upper), and F2 (lower)

generations. The central line is the mean, the box is the standard deviation and the whisker is the standard error of the

vitellogenin expression. o,p'-DDT F1 Subadult Vitellogenin CERI MMT

Concentration (ug/L)Vitellogenin Protein (ng/mg liver)

0 0.032 0.07 0.22 0.69 1.9 0 1000 2000 3000 4000 5000 6000 7000 8000

XY

XX



# * *

*

* *

o,p'-DDT F2 Subadult Vitellogenin CERI MMT

Concentration (ug/L)Vitellogenin Protein (ng/mg liver)

0 0.032 0.07 0.22 0.69 1.9 0 1000 2000 3000 4000 5000 6000 7000 8000

XY

XX



# *

* *

*

*Figure 5, subadult secondary sex characters. XY secondary sex characters (SSC) of sub adult medaka in F1 (upper)

and F2 (lower ) generations after exposure to o,p’

- DDT . The central line is the mean, the box is the standard deviation and

the whisker is the standard error of the number of anal fin papillae. * = significantly different from controls. o,p'-DDT F2 Subadult (XY only) SSC CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 20 40 60 80 100Anal Fin Papillae (#/fin)



# * o,p'-DDT F1 Subadult (XY only) SSC CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 20 40 60 80 100Anal Fin Papillae (#/fin)



# *Figure 6, subadult gonad phenotype. Gonad phenotype of XY subadult medaka in F1 (upper) and F2 (lower) after

exposure to o,p’

- DDT . The stacked bar graphs show the percent of individuals with testis, intersex, or ovary. The

number in the boxes is the number of individuals in each treatment wit h that gonad phenotype . Intersex gonad is defined

as a single gonad with any amount of both ovarian and testicular tissue with no measurement of relative proportion.Figure 7, subadult weight. Weight of subadult medaka in F1 (upper) and F2 (lower) generations after exposure to o, p ’

- DDT. The central line is the mean, the box is the standard deviation and the whisker is the standard error of the weight

measured in mg. Weights were reported sans genotypic or phenotypic data. o, p-DDT F1 Subadult Weight CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 50 100 150 200 250 300 350 400Body Weight (mg)

o, p-DDT F2 Subadult Weight CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 50 100 150 200 250 300 350 400Body Weight (mg)Figure 8, subadult length. Length of subadult medaka in F1 (upper) and F2 (lower) generations after exposure to o, p ’

- DDT. The central line is the mean, the box is the standard deviation a nd the whisker is the standard error of the length

measured in mm. o, p-DDT F1 Subadult Length CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 5 10 15 20 25 30 35Length (mm)

o, p-DDT F2 Subadult Length CERI MMT

0 0.032 0.07 0.22 0.69 1.9

Concentration (ug/L) 0 5 10 15 20 25 30 35Length (mm) DTable 2. Subadult (8 wph) survival.

Subadult Survival (mean % ± S.D.)

Treatment

Generation control 0.032 μg/L 0.07 μg/L 0.22 μg/L 0.69 μg/L 1.9 μg/L

F1 100 100 96.7±5 100 100 100

F2 100 100 98±4 100 100 100

Table 3. Hatch.

Hatch (mean % ± S.D.)

Treatment

Generation control 0.032 μg/L 0.07 μg/L 0.22 μg/L 0.69 μg/L 1.9 μg/L

F1 99±2 88±27 100 98±3 97±6 99±0.1

F2 95±9 100 91±14 99±2 68±43 92±12Test Chemical: 4

- chloro

- 3

- methylphenol (CMP)

MOA: in vitro ER agonist (rainbow trout)

Protocol: full research MMT

Lab: MED

Summary of Test Chemical and MOA:

CMP is a chlorophenol used, amongst other things, as an antiseptic and disinfectant in cosmetics.

It has been detected in wastewater and sediment, and has been shown to be toxic to fish and aquatic

invertebrates. In work done at MED, CMP binds to both re combinant human and rainbow trout ERs and

induces rainbow trout vitellogenin mRNA at about the same concentrations as overt toxicity. In in vitro

cell assays, CMP acted as an ER agonist at about the same concentration as its toxic activity.

Summary of P rotocol:

The MMT completed by USEPA

- MED t esting the effects of CMP was done with the full

research MMT protocol that has been detailed previously (pp. ). Briefly, the CMP MMT protocol

spanned 2 1⁄2 generations, in which in the F0 generation chemical expos ure was initiated with spawning

pairs at the beginning of 1 0 wpf (69 dpf) and continues through the beginning o f 15 wpf (105 dpf) at

which time the fish were sacrificed. In the F1 and F2 generations, fish were continuously exposed from

embryo through adul t spawning. Fish were sampled at two major life

- stages; subadults (in the 8 th wpf ;

50

- 51 dpf) in the F1 and F2 generations, and mature adults (at the start of the 15 th wpf ; 105 dpf ) in F0,

F1, and F2. Reproduction was evaluated for 4 consecutive days pe r week over 5 weeks from 10 wpf to 15

wpf.

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The

proportionate chemical concentration diffe rence between treatments was 0.5 . The average measured CMP

concentrations in the test were 2 1, 43, 88, 165, and 345 μ g/L. Reproduction in all three generations (F0,

F1, and F2) consisted, nominally, of 6 breeding pairs per concentration and 6 breeding pairs for controls.

These breeding pairs were also the fish sampled as adults. At the subadult (8 wpf; 50

- 51 dpf) sampling, 8

of the 10 fish per aquaria were randomly sacrificed to a) produce the subadult samples an d to b) leave

behind the breeding pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair (4

consecutive days per week over 5 weeks) in F0, F1, and F2; in adults in F0, F1, and F2, leng th, weight,

VTG gene expression, SSC, and histopathology evaluation of gonad, kidney, and liver were recorded; and

in subadults in F1 and F2, VTG gene expression, SSC, gonad histology, weight and length were recorded.

In addition, hatch and survival from hatch to 3 wpf, 3 wpf to 8 wpf, and 9 wpf to 16 wpf were recorded.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with both the direction of the effect, and the

statistical test used indicated. The LOEC is color

- coded with more intense colors for lo wer

concentrations of CMP

. Apart from some isolated effects, exposure to CMP did not appear to have a

negative impact on the medaka of this MMT.

In a separate study, the lethal concentration of CMP was determined to verify th at the MMT was

run with CMP concentrations that would be just below toxic and therefore, demonstrate an EDC effect if

one was present. Starting with 1 dph medaka, the 14 day LC50 of CMP was determined to be 1090 μ g/L

, but at concentrations up to 1200 μ g/L , there was no vitellogenin gene induction. Therefore, it appearsthat the MMT was indeed performed on concentrations of CMP that were close to toxic, but CMP was not

acting as an EDC in the MMT.

Reproduction (Figure 1) :

No effects were observed on rep roduction in the CMP MMT at any concentration or in any

generation.

Adult Vitellogenin (Figure 2) :

In the F0, F1, and F1 adult samples, the effect of CMP exposure on vitellogenin gene expression

(vitellogenin mRNA copy number per ng total RNA) was negli gible. In F1 and F2, there were no

significant changes in expression in either XX or XY fish. As determine by the Jonckheere

- Terpstra

trend test, there was an increase in expression starting at 88 μg/L CMP. However the increased copy

number amounted to a 2.6 times that of controls at the responses highest in the 345 μg/L treatment and

less than 2 times that of controls at the LOEC of 88 μg/L. Not only is the magnitude of the effect

minimal, the biological consequence of slightly increased vitellogenin i n the liver of XX fish is not clear.

Adult Secondary Sex Character s (Figure 3) :

No effects were observed on SSC in the CMP MMT at any concentration or in any generation.

Adult Gonad phenotype (no figure) :

Gonad phenotypes were simply one of three c ategories: testis, ovary, and intersex with any

number of testicular oocytes putting an individual in the intersex category. There were no gonad

phenotypes that did not match the determined gender genotype in the adults of the three generations. In

addit ion, there were no instances of intersex gonads observed in any of the adults as well.

Adult Pathology (Tables 2

– 3) :

There were only a few pathologies observed in the CMP MMT. In adult XY fish across all

generations, the only pathology that was statistically significant at least at as a mild severity (2) was

vacuolization of the kidney tubule epithelium in F2 only at a LOEC of 21 μg/L. All other observed

pathologies were minimal severity (1) at 165 μg/L or higher except mineralization in the testis which had

a LOEC of 43 μg/L. In XY fish across the three generations, there were no consistent pathological

findings.

In XX adult fish, no pathologies were significant above minimal severity (1) or with LOECs

lower than 165 μg/L. In addition, no pathologies were significant in XX fish in F2. As with the XY fish,

no consistent findings were observed across the generation s.

Adult Weight (Figure 4) :

No effects were observed on body weight in the CMP MMT at any concentration or in any

generation.

Adult Length (no figure) :

No effects were observed on body weight in the CMP MMT at any concentration or in any

generation.Adult Survival (Table 4) :

No effects on adult survival were observed in the CMP MMT at any concentration or in any

generation.

Subadult Vitellogenin (Figure 5) :

No effects on vitellogenin expression in either XX or XY fish were observed in the CMP MM T

in F1. The highest concentration of CMP (345 μg/L) in F2 did lower vitellogenin expression in XX fish

slightly; there was 1/5 th the expression as compared to XX controls. In addition, the XX fish exposed

to165 μg/L CMP tended to have lower expression t han controls that was not statistically significant. The

significance at 345 μg/L CMP was only with the Jonckheere

- Terpstra test which was strengthened by

both 165 and 345 μg/L trending lower than controls. The decrease in vitellogenin expression did not

result in a measured decrease in reproduction or any other of the measured endpoints. No alterations in

expression were observed in XY fish.

Subadult Secondary Sex Characters (Figure 6) :

No effects on adult survival were observed in the CMP MMT at any concentration or in any

generation.

Subadult Gonad Phenotype (no figure) :

There were no gonad phenotypes that did not match the determined gender genotype in the

subadults of the two generations. In addition, there were no instances of intersex gonads observed in any

of the subadults as well.

Subadult Pathology (Tables 5

– 6) :

An interpretation of the results for the subadults for the CMP MMT was that no EDC

- related

pathologies were observed; only pathologies associated with non

- EDC toxicity at lower severity were

observed. For XY subadults, F1 was about the same as F2. In F1, the LOEC was 345 μg/L for all

observed pathologies including kidney tubule dilatation with severity of 2 and increased kidney

hematopoietic tissue, kidney tubule minerali zation, and cystic liver degeneration all at severity of 1. The

F2 pathologies were the same as F1 with a lower LOEC (88 μg/L CMP) for both kidney tubule dilatation

and kidney tubule mineralization.

The LOEC in F1, XX subadults was 345 μg/L (increased h ematopoietic tissue in the kidney,

kidney tubule mineralization, and cystic degeneration in the liver) except for kidney tubule dilatation

which was at 43 μg/L. The only pathology that was significant in F2 XX subadults was increased

hepatocellular vacuol ization at a severity of 1 with a LOEC of 345 μg/L.

Sub a dult Weight (Figure 7) :

In F1, XY fish at 165 μg/L and higher in the CMP MMT weighed less than the controls. There

was a relatively small decrease (210 mg vs. 185 mg) in the two highest treatments that was only

significant with the Jonckheere

- Terpstra test and not the others implemented within StatCHARRM S.

While not a particularly large response, it is also present at the same LOEC (165 μg/L) for the other

growth measurement: length. This effect was not observed in the F2 generation. Also, no changes in

weight were present in the XX fish of either gene ration.Subadult Length (Figure 8) :

The pattern for the length growth measurement was identical to that of weight: a decrease, in this

case shorter fish, in F1 XY fish starting at 165 μg/L that was not observed in XX fish or in XY fish from

F2. Again t he absolute decrease was minimal, 27 mm in controls vs. 25 mm at 345 μg/L CMP

.

Subadult Survival (Table 7

- 8) :

CMP did not have a negative effect on survival from either hatch to 3 wpf or from 4 wpf to 8 wpf.

Subadult Hatch (Table 9) :

CMP did not ha ve a negative effect on hatch in either F1 or F2.Table 1. Summary table of LOECs for the various biological data collected during the MED CMP MMT.

CMP MMT LOEC Summary CMP (ug/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

Subadult (8 wpf) Subadult (8 wpf) Adult (16 wpf) Adult (16 wpf) Adult (16 wpf) 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 21 5

Weight JT:165 ↓ LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 3 43 14

Length JT:165 ↓ LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 4 88 28

Survival LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 5 165 22

Hatch LOEC > 345 LOEC > 345 6 345 74

Papillae LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345

Vtg LOEC > 345 LOEC > 345 LOEC > 345 JT:345 ↓ LOEC > 345 JT:88 ↑ LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345

Gonad Phenotype LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345 LOEC > 345

Fecundity LOEC > 345 LOEC > 345 LOEC > 345

Fertility LOEC > 345 LOEC > 345 LOEC > 345

Highest Conc 345 345 345 345Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviat ion,

and the whisker is the standard error of the number of eggs spawned per

day by each replicate. CMP F1 Reproduction MED MMT

0 21 43 88 165 345

Concentration (ug/L) 0 10 20 30 40 50

Fecundity

Fertility CMP F2 Reproduction MED MMT

0 21 43 88 165 345

Concentration (ug/L) 0 10 20 30 40 50

Fecundity

Fertility

CMP F0 Reproduction MED MMT

0 21 43 88 165 345

Concentration (ug/L) 0 10 20 30 40 50

Fecundity

Fertility

Raw DataFigure 2. Gender specific vitellogenin gene expression of adult (16 wpf)

medaka in F1 (upper left), F2 (upper right), and F0 (lower left) generations

after exposed to CMP. The central line is the mean, the box is the standard

deviation and the whisker is the stan dard error of the vitellogenin expression.

Vitellogenin gene expression is graphed relative to the gender specific

controls. JT = Jonckheere

- Terpsta; Du = modified Dunn’s. CMP F1 Adult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 21 43 88 165 345 -2 0 2 4 6 8 10 12 14

XY

XX CMP F2 Adult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 21 43 88 165 345 -4 -2 0 2 4 6 8 10 12 14 16 18 20

XY

XX

CMP F0 Adult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 21 43 88 165 345 0 2 4 6 8 10

XY

XX

JT Du JT

JTFigure 3. Gender specific secondary sex characters (SSC) of adult (16 wpf)

medaka in F1 (upper left), F2 (upper right), and F0 (lower left) generations

after exposure to CMP. The central line is the mean, the box is the standard

deviation and the whisker is the s tandard error of the number of anal fin

papillae, that is the SSC. CMP F1 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 21 43 88 165 345 0 20 40 60 80 100 120 140 160 180

XY

XX

CMP F0 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 21 43 88 165 345 0 20 40 60 80 100 120 140 160 180 200 220 240

XY

XX

Individual Fish CMP F2 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 21 43 88 165 345 -20

0 20 40 60 80 100 120 140 160 180

XY

XX

### Table 2. Significant pathologies as indicated by RSCABS in adult XY medaka of the CMP MED MMT

F0 XY F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Increased Spermatogonia 345 μg/L 1 345 μg/L 1

- - - - 345 μg/L 1 345 μg/L 1

Interstitial Cell Hyperplasia

Hypertrophy 345 μg/L 1 345 μg/L 1 165 μg/L 1 16 5 μg/L 1

- - - -

Mineralization

- - - - 165 μg/L 1 16 5 μg/L 1 43 μg/L 1 43 μg/L 1

KIDNEY Tub Epith Vacuolization

- - - - - - - - 21 μg/L 2 21 μg/L 2

Tubule Mineralization

- - - - - - - - 345 μg/L 1 345 μg/L 1

LIVER Vacuolated Foci

- - - - 345 μg/L 1 345 μg/L 1

- - - -



### Table 3. Significant pathologies as indicated by RSCABS in adult XX medaka of the CMP MED MMT

F0 XX F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

KIDNEY Tubule Dilatation

- - - - 165 μg/L 1 165 μg/L 1

- - - - Tubule Regeneration

- - - - 165 μg/L 1 165 μg/L 1

- - - -

LIVER Cystic Degeneration 165 μg/L 1 165 μg/L 1

- - - - - - - -

Vacuolated Foci

- - - - 345 μg/L 1 345 μg/L 1

- - - -Figure 4. Gender specific weight of adult (16 wpf) medaka in F1 (upper

left), F2 (upper right), and F0 (lower left) generations after exposed to CMP.

The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the weigh t measured in mg. CMP F1 Adult Weight MED MMT

Concentration (ug/L)Body Weight

0 21 43 88 165 345 0 200 400 600 800 1000 1200 1400 CMP F2 Adult Weight MED MMT

Concentration (ug/L)Body Weight

0 21 43 88 165 345 0 100 200 300 400 500 600 700 800 900

CMP F0 Adult Weight MED MMT

Concentration (ug/L)Body Weight

0 21 43 88 165 345 0 100 200 300 400 500 600 700 800 XY

XXFigure 5

. Gender specific vitellogenin gene expression of subadult (8 wpf)

medaka in F1 (upper), and F2 (lower) g enerations after exposed to CMP

. The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the vitellogenin expression. Vitellogenin

gene expression is graphed relative to the gender specific controls. JT =

Jonckheere

- Terpstra trend test CMP F1 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 21 43 88 165 345 0 10 20 30 40 50

XY

XX CMP F2 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 21 43 88 165 345 0 10 20 30 40 50 XY

XX

JTFigure 6

. Gender specific secondary sex characters (SSC) of subadult (8

wpf) medaka in F1 (upper), and F2 (lower) generations after exposure to

CMP. The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the number of anal fin papillae that is the

SSC. CMP F1 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 21 43 88 165 345 0 20 40 60 80 100 120 140 160

XY

XX CMP F2 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 21 43 88 165 345 0 20 40 60 80 100 120 140 160

XY

XXTable 4

. Significant pathologies as indicated by RSCABS in subadult XY medaka of the CMP MED MMT

F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

KIDNEY Increased hematopoietic tissue 345 μg/L 1 345 μg/L 1 345 μg/L 1 345 μg/L 1

Tubule Dilatation 345 μg/L 2 345 μg/L 2 88 μg/L 1 345 μg/L 2

Tubule Mineralization 345 μg/L 1 345 μg/L 1 88 μg/L 1 88 μg/L 1

LIVER Cystic degeneration 345 μg/L 1 345 μg/L 1 345 μg/L 1 345 μg/L 1

Table 5

. Significant pathologies as indicated by RSCABS in subadult XX medaka of the CMP MED MMT

F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Proteinaceous Fluid 345 μg/L 1 345 μg/L 1

- - - -

LIVER Increased Hepatocell Vacuol

- - - - 345 μg/L 1 345 μg/L 1

KIDNEY Nephropathy 345 μg/L 1 345 μg/L 1

- - - -

Proteinaceous Fluid 345 μg/L 2 345 μg/L 2

- - - -

Tubule Dilatation 43 μg/L 1 43 μg/L 1

- - - -

Tubule Mineralization 165 μg/L 1 165 μg/L 1

- - - -Figure 7

. Gender specific weight of subadult (8 wpf) medaka in F1 (upper)

and F2 (lower) generations after exposure to CMP. The central line is the

mean, the box is the standard deviation and the whisker is the standard error

of the weight measured in mg. JT = Jonckheere

- Terpstra trend test. CMP F1 Subadult Weight MED MMT

Concentration (ug/L)Body Weight

0 21 43 88 165 345 0 50 100 150 200 250 300 350 400

XY

XX

JT JT CMP F2 Subadult Weight MED MMT

Concentration (ug/L)Body Weight

0 21 43 88 165 345 0 50 100 150 200 250 300 350 400

XY

XXFigure 8

. Gender specific length of subadult (8 wpf) medaka in the F1

ge neration after exposure to CMP. The central line is the mean, the box is

the standard deviation and the whisker is the standard error of the length

measured in mm. JT = Jonckheere

- Terpstra tre nd test. CMP F1 Subadult Length MED MMT

Concentration (ug/L)Length

0 21 43 88 165 345 0 5 10 15 20 25 30

XY

XX JT JTTable 6. Early life (4 wpf) survival.

Early Life Survival (mean %)

Treatment

Generation control 21 μg/L 43 μg/L 88 μg/L 165 μg/L 345 μg/L

F1 missing missing missing missing missing missing

F2 65 93 94 90 68 73

Table 7. Subadult (8 wpf) survival.

Table 8. Hatch.

Hatch (mean %)

Treatment

Generation control 21 μg/L 43 μg/L 88 μg/L 165 μg/L 345 μg/L

F1 77 87 94 96 87 86

F2 68 81 79 94 90 95 Subadult Survival (mean %)

Treatment

Generation control 21 μg/L 43 μg/L 88 μg/L 165 μg/L 345 μg/L

F1 98 100 93 97 92 90

F2 95 97 97 95 98 93Test Chemical: tamoxifen

MOA: mixed ER agonist/antagonist or selective ER modulator

Protocol: abbreviated MMT

Lab: NIES (Japan)

Summary of Test Chemical and MOA:

Tamoxifen is classified as a selective ER modulator (SERM) as it has multiple actions both as a

ER antagonist and in specific tissues/species as a ER agonist. It is a pharmaceutical used, because of its

ER antagonistic properties, in the treatment of breast cancer, and is often prescribed to postmenopausal

women. After binding to the ER, tamoxifen has been shown to act as both agonist and antagonist in

various species including the medaka, in which it increased plasma vitellogenin in males but decreased

plasma vitellogenin in females. In addition, tamoxifen (at 625 μ g/L) reduced the hatch rate and the

fecundity of medaka.

Summary of Protocol:

The MMT completed by NIES in Japan testing the effects of tamoxifen was done with an

abbreviated MMT protocol that has been detailed previously. Briefly, the tamoxifen MMT protocol

involved three separ ate generations of medaka in which in the F0 generation chemical exposure was

initiated with adult spawning pairs for and lasted for four weeks. In the F1 generation, fish were

continuously exposed from embryo through adult spawning while in F2 fish were reared again with

continuous exposure to 65 dpf

. In F0, no biological data was collected as this generation served only to

“load” gametes with tamoxifen. In F1, sampling of the fish was done at 54

- 55 dpf (subadult stage) and

again at 92

- 93 dpf (adult sta ge). Also in the F1 generation, reproduction was monitored every day for

three weeks from 11 to 13 wpf. In F2, a fish were sampled at a single time, 65

- 66 dpf, for various

endpoints, but no reproductive assessment was made in F2.

The general design e mployed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The

proportionate chemical concentration difference between treatments was 0.5. The average measured

tamoxifen concentrations in the test were 1.3, 2.5, 5, 10, and 20 μ g/L. Reproduction in F1 consisted,

nominally, of 6 breeding pairs per concentration and 6 breeding pairs for controls. These breeding pairs

were also the fish sampled as adults. At the F1 subadult (8 wpf; 54

- 55 dpf) sampling, 8 of the 10 fish per

aquaria were randomly sacrificed to a) produce the subadult samples and to b) leave behind the breeding

pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair (21

consecutive days) in F1; in adults in F1, hepatic vitellogenin and SSC were recorded; in F1, weight and

length, gonad phenotype, SSC, and hepatic vitellogenin were recorded; and finally in F2 su badults,

weight and length, gonad phenotype, SSC, and hepatic vitellogenin were recorded. In addition, hatch in

F1 and F2 was recorded.

Summary of Results (Table 1):

The LOECs are summarized in a “heat table” with the direction of the effect indicated. The

LOEC is color

- coded with more intense colors for lower concentrations of tamoxifen , and the direction of

the change from controls is indicated by an up or down arrow

.

The LOEC for reproductive effects was 20 μ g/L in which tamoxifen reduced fecundit y

substantially. Even though the numbers of offspring were greatly reduced at this concentration, therewere enough to continue the treatment group into the F2 generations. The EDC

- related endpoints, SSC

and vitellogenin, tended to have LOECs of 1.3 μ g/L in subadults and slightly higher (2.5

– 5 μ g/L) in

adults. There was a large discrepancy between LOECs of F1 and F2 subadult in the SSC endpoint, but

this and other more nuanced differences between these two generations are explained by the

developmental stage the fish were at when the sampling was done. Simply put there was a big difference

between fish at 54 dpf in F1 and 65 dpf in F2 so that in reality the two generations of subadult samplings

are not directly comparable. Often this was seen in much different values in control fish of the two

generations.

Tamoxifen can act as either an ER agonist or an antagonist depending on species and tissue. The

results of this MMT are consistent with this MOA. For instance, hepatic vitellogenin concentrati ons in

males were elevated with tamoxifen exposure but in females, vitellogenin was decreased at the same

concentrations of tamoxifen. Tamoxifen appears to bind to the ER in both males and females. In males,

the binding is able to activate various estro gen

- ER cascades leading to results like vitellogenin

production. In females, the tamoxifen

- ER complex is less effective at activating these cascades;

therefore, tamoxifen reduces the levels of their end

- products such as vitellogenin.

Reproduction (Figu re 1):

Reproduction was reduced upon exposure to 20 μ g/L tamoxifen, the highest concentration tested.

Specifically, fecundity was reduced while the percentage of fertile eggs was not altered by any of the

concentrations of tamoxifen tested. Reduced fec undity was present in all the weeks of the assessment.

Adult Vitellogenin (Figure 2):

Tamoxifen caused production of vitellogenin in the livers of F1 adult males at 2.5 μ g/L and

above. The increase was modest in severity: 2.5 times, 2 times, 2.9 times and 2.9 times as much

vitellogenin in the 2.5 μ g/L, 5 μ g/L, 10 μ g/L, and 20 μ g/L treatments, respectively, over that of control

males. In addition, tamoxifen reduced the conce ntration of hepatic vitellogenin in the 5 μ g/L , 10 μ g/L,

and 20 μ g/L treatment by 0.5 times, 0.8 times, and 0.7 times, respectively. This type of response is not

surprising for a mixed ER agonist/antagonist like tamoxifen. Upon binding to the ER, tt see ms likely that

tamoxifen is less able to cause the downstream cascade than the native ligand. Therefore, tamoxifen can

cause an increase in vitellogenin production in males where circulating estrogen is very low while

actually inhibiting estrogen mediated processes like vitellogenin synthesis in females.

Adult Secondary Sex Characters (Figure 3):

In F1, tamoxifen increased the number of anal fin papillae, the measure of secondary sex

characters in medaka, in XY fish . The increase was from a mean of 106 in control males to 151 papillae

in the 5 μ g/L treatment, 136 papillae in the 10 μ g/L treatment, and 140 papillae in the 20 μ g/L treatment.

It is unclear by what MOA tamoxifen would cause an increase in SSC, and in f act, tamoxifen reduced the

number of papillae in F1 and F2 male subadults; a much more intuitive response of a ER agonist.

Adult Gonad phenotype:

No gonad phenotype data was reported for adult fish.Adult Pathology:

No pathology data was reported for adult fish.

Adult Weight:

No weight data was reported for adult fish.

Adult Length:

No length data was reported for adult fish.

Adult Survival:

No length data was reported for adult fish.

Subadult Vitellogenin (Figure 4):

In subadult fish, tamoxifen caused significant increases in vitellogenin of male fish in the 1.3

μg/L through 20 μg/L treatment groups (all the concentrations that were tested) of the F1 generation. The

increased hepatic vitellogenin concentration ranged from 2.7 times gr eater than in controls at the 1.3 μ g/L

treatment to 18 times greater in the 20 μ g/L treatment. A very similar response was observed in the F2

subadults: the LOEC again was 1.3 μ g/L and the range of the actual increase was from 1.8 times to 21

times that o f control males. In addition, the concentration of hepatic vitellogenin decreased in females,

starting at 1.3 μ g/L tamoxifen. The amount of the decrease ranged from 0.4 to 0.1 times that of controls

trending from low to high tamoxifen concentrations.

Qualitatively, the response seen in the F2 fish is the same as observed in the adult fish of F1,

increased vitellogenin in males and decreased in females. Vitellogenin did not decrease in females of F1

presumably because vitellogenin production in the li ver was low, almost the same as males, especially as

compared to F2 subadult or adult females. At 54 dpf, the sampling time in F1, the females appear to have

just started to ramp up vitellogenin production, and therefore, seem to be less sensitive to the ER

antagonist effects of tamoxifen.

Subadult Secondary Sex Characters (Figure 5):

In F1 subadults (54 dpf), the only effect observed with tamoxifen exposure was decrease in

papillae in the males of the 20 μ g/L treatment. However, there many males a cross all the treatments and

controls that had either no or very few papillae, and the mean control number in males was only 15 which

decreases the statistical power of this dataset. On the other hand, the subadults in F2, which were

sampled 11 days later than they were in F1, had much more papillae in controls (80) and the LOEC was

also much lower (1.3 μ g/L). The developmental difference between the subadults in F1 compared to F2

is large which does explain the difference in the LOECs observed both becau se of the much higher

control mean and its greater statistical power, and because the fish are more able to respond biologically.

Possibly tamoxifen, as an ER agonist, was able to slightly feminize the male fish in F2 by 65 dpf but was

not able to have th e same effect in F1 at 54 dpf.

Subadult Gonad Phenotype (Table 2

- 3):Because of limitations with the data reported by NIES, statistical analysis was not done and firm

conclusion regarding effect concentrations are difficult to make. The data from both generations is

suggestive of tamoxifen delaying or inhibiting the maturation of testes. However, this interpretation is

based solely on the two tables provided, it is primarily qualitative, and therefore the confidence that this is

a correct conclus ion is relatively low.

Subadult Pathology:

No pathology data was reported for subadult fish.

Subdult Weight (Figure 6):

In both generations, the subadult females exposed to 20 μ g/L weighed less than the control

females: 0.13 g compared to 0.09 g or about a 30% decrease in F1 and 0.24 g compared to 0.19 g or a

20% decrease in F2 with a tamoxifen exposure of 20 μ g/L. Male weight was reduced in F1 by about 30%

(0.13 g compared to 0.09 g), while in F2, male weight was unaffected by tamoxifen.

Subadult Length (Figure 7):

In F1 subadults, only the length of males was decreased at a LOEC of 20 μ g/L. The decrease was

by about 16%, 22.8 mm in controls compared to 19.3 mm in the 20 μ g/L treatment. The females were

unchanged with tamoxifen exposure in F1. On the other hand, exposed males in the F2 subadults

(sampled at 65 dpf) were the same length as controls while females in the 20 μ g/L treatment were slightly

shorter than control: 27.9 mm in controls versus 26.3 mm in the 20 μ g/L treatment.

Subadult Survival:

No survival data was reported for subadult fish.

Subadult Hatch (Table 4):

While there were no statistically significant reductions in hatch in any of the treatments in either

generation, there did appear to be a trend from the normal high hatch rate in controls to lower hatch rates

with increasing tamoxifen concentrations. For instance, F1 controls hatched at a rate of 95% while in the

20 μ g/L treatment, the hatch ra te was 72%. Likewise, F2 controls had 85% hatch while the 10 and 20

μ g/L treatments had hatch rates of 57% and 68%, respectively.Table 1. Summary table of LOECs for the various biological data collected during the NIES Tamoxifen MMT.



## Tamoxifen MMT LOEC Summary Tamoxifen (ug/L)

F1 F2 F0 F1 Trt Conc S.D.

subadult (54 dpf) subadult (65 dpf) adult adult (92 dpf) 1 0 0

XY XX XY XX XY XX XY XX 2 1.3 0.19

Weight 20↓ 20↓ LOEC>20 

## 20↓ 3 2.5 0.28

Length 20↓ LOEC>20 LOEC>20 

## 20↓ 4 5 0.7

Survival 5 10 1.3

Hatch LOEC>20 LOEC>20 6 20 2.6

SSC 20↓ LOEC>20 

## 1.3↓ LOEC>20 

## 5↑ LOEC>20



## Vtg 1.3↑ LOEC>20 

## 1.3↑ 1.3↓ 2.5↑ 5↓

Gonad Phenotype unknow n unknown unknown unknown



## Fecundity 20↓

Fertility 20↓

Highest Conc 20 20 20 20

LOECs are color coded with more intense colors for lower concentrations. The direction of the change from controls is indica ted by the direction of the arrow.Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 generation. The central line is the mean, the box is the

standard deviation, and the whisker is the standard error of the number of

eggs spawned per day by each replicate. Tamoxifen F1 Reproduction NIES MMT

0 1.3 2.5 5.1 10 20

Concentration (ug/L) 0 5 10 15 20 25 30 35

Fecundity

Fertility 

## *

*Figure 2, adult vitellogenin. Gender specific hepatic vitellogenin of adult

medaka in F1. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the hepatic vitellogenin

concentra tion. Significance is indicated by a letter: JT = Jonckheere

- Terpstra

and D = Dunnett’s. Tamoxifen F1 Adult Vitellogenin NIES MMT

Concentration (ug/L)Vtg (ng/mg liver)

0 1.3 2.5 5 10 20 0 200 400 600 800 1000

D JT JT JT JT

JT JT JT DFigure 3, adult secondary sex characters. Gender specific secondary sex

characters (SSC) of adult (14 wpf) medaka in F1 after exposure to tamoxifen.

The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the number of anal fin papillae that is the

SSC. D or JT = significantly different from controls as determined by

Dunnett’s test or Jonckheere

- Terpstra trend test, respectively. Tamoxifen F1 Adult (92 dpf) SSC NIES MMT

0 1.3 2.5 5 10 20

Concentration (ug/L) 0 20 40 60 80 100 120 140 160 180Anal Fin Papillae (#/fin) D

JT JT

JTFigure 4, subadult vitellogenin. Gender specific hepatic vitellogenin of

adult medaka in F1. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the hepatic vitellogenin

concentration. Significance is indicated by a letter: JT = Jonckheere

Terpstra, TD = Tamhane

- Dunnett, and Du = Dunn. Tamoxifen F1 Subadult (54 dpf) Vitellogenin NIES MMT

Concentration (ug/L)Vtg (ng/mg liver)

0 1.3 2.5 5 10 20 -50

0 50 100 150 200 250 300 350 400

Du

Du Du Du Du Du Tamoxifen F2 Subadult (65 dpf) Vitellogenin NIES MMT

Concentration (ug/L)Vtg (ng/mg liver)

0 1.3 2.5 5 10 20 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

TD

Du JT JT JT JT JT Du Du Du TD TD TD

TDFigure 5, subadult secondary sex characters. Gender specific secondary

sex characters (SSC) of sub adult medaka in F1 (left), and F2 (right )

generations after exposure to tamoxifen . The central line is the mean, the

box is the standard deviation and the whisker is the standard error of the

number of anal fin papillae that is the SSC. D, Du or JT = significantly

different from controls as determined by a Dunnett test, Dunn’s test or

Jonckheere

- Terpstra trend test, respectively Tamoxifen F1 Subadult (54 dpf) SSC NIES MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 1.3 2.5 5 10 20 0 10 20 30 40 50 60

XY

XX

Du Tamoxifen F2 Subadult (65 dpf) SSC NIES MMT

0 1.3 2.5 5 10 20

Concentration (ug/L) 0 20 40 60 80 100 120 140 160Anal Fin Papillae (#/fin)

D JT D

D DTable 2. Gonad phenotype of F1 subadult (54 dpf), XY and XX medaka

treatment

( μ g/L) genetic sex number of

medaka gonad

testis immature ovary

control XY 26 12

35

XX 22 1

1. 3 XY 27 7

39

XX 20 1

2.5 XY 21 16

32

XX 27 0

5 XY 21 6

38

XX 24 1

10 XY 28 5

43

XX 20 0

20 XY 15 0

38

XX 24 1 Table 3. Gonad phenotypce of F2 subadult (65 dpf), XYand XX medaka

treatment

( μ g/L) genetic

sex N of male

medaka gonad

testis immature ovary

control XY 26 26

0

XX 31 31

1. 3 XY 32 31

6

XX 28 23

2.5 XY 32 30

12

XX 28 18

5 XY 36 38

4

XX 25 19

10 XY 36 29

11

XX 24 20

20 XY 15 17

2

XX 15 11Figure 6, subadult weight. Gender specific weight of sub adult medaka in

F1 at 54 dpf (left), and F2 at 65 dpf (right) after exposure to tamoxifen . The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the weight

. Significantly different from controls is

indicated by D = Dunnett test, TD = Tamhane

- Dunnett, JT = Jonckheere

Terpstra trend test, and Du = Dunn

. Tamoxifen F1 Subadult (54 dpf) Weight NIES MMT

Concentration (ug/L)Weight (g)

0 1.3 2.5 5 10 20 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

XX

XY Du TD

JT JT Tamoxifen F2 Subadult (65 dpf) Weight NIES MMT

Concentration (ug/L)Weight (g)

0 1.3 2.5 5 10 20 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

XX

XY

TD JTFigure 7, subadult length. Gender specific length of sub adult medaka in F1

at 54 dpf (left), and F2 at 65 dpf (right) after exposure to tamoxifen . The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the length (mm)

. Significantly different from controls is

indicated by JT = Jonckheere

- Terpstra trend test, and Du = Dunn

. Tamoxifen F1 Subadult (54 dpf) Length NIES MMT

Concentration (ug/L)Length (mm)

0 1.3 2.5 5 10 20 0 2 4 6 8 10 12 14 16 18 20 22 24 26

XX

XY Du Tamoxifen F2 Subadult (65 dpf) Length NIES MMT

Concentration (ug/L)Length (mm)

0 1.3 2.5 5 10 20 0 5 10 15 20 25 30

XX

XY Du JTTable 4. Hatch

Hatch (mean % )

Treatment

Generation control 1.3 μg/L 2.5 μg/L 5 μg/L 10 μg/L 20 μg/L

F1 95 95 86 85 80 72

F2 85 75 64 65 57 68Test Chemical: 17β

- trenbolone

MOA: androgen (androgen receptor agonist)

Protocol: research MMT

Lab: MED

Summary of Test Chemical and MOA:

17β

- trenbolone is a metabolite of trenbolone acetate, an anabolic steroid used to promote muscle

mass in the beef cattle industry. 17β

- tren bolone acts as a potent agonist of androgen receptors in a variety

of systems including fish where it has been shown to reduce fecundity, vitellogenin, and generally induce

masculinization of both external secondary sex characteristics and gonad phenotypes . In fathead minnow,

the LOEC for decreased vitellogenin in females, increased secondary sex characters in males, and

decreased fecundity were all 50 ng/L in a 21

- day exposure. In a similar 21

- day exposure of medaka, the

LOEC for changes in secondary sex characters and decreased vitellogenin was 365 ng/L and 39.7 ng/L,

respectively. Trenbolone is both a model androgen with 10 fold greater affinity for the fathead AR than

testosterone, and a chemical of environmental concern.

Summary of Protocol:

The MMT completed by USEPA

- MED testing the effects of trenbolone was done with the full

research MMT protocol that has been detailed previously (pp. ). Briefly, the trenbolone MMT protocol

spanned 2 1⁄2 generations, in which in the F0 generation chemical ex posure was initiated with spawning

pairs at the beginning of 11 wpf (76 dpf) and continues through the beginning of 16 wpf (111 dpf) at

which time the fish were sacrificed. In the F1 and F2 generations, fish were continuously exposed from

embryo through a dult spawning. Fish were sampled at two major life

- stages; subadults (in the 8th wpf;

50

- 51 dpf) in the F1 and F2 generations, and mature adults (at the start of the 16th wpf ; 111 dpf ) in F0,

F1, and F2. Reproduction was evaluated for 4 consecutive days per week over 5 weeks from 11 wpf to 16

wpf.

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The

proportionate chemical concentration difference between treatments was 0.4. The average measured

t renbolone concentrations in the test were 2, 5, 13, 32, and 84 ng/L. Reproduction in all three generations

(F0, F1, and F2) consisted, nominal ly, of 6 breeding pairs per concentration and 6 breeding pairs for

controls. These breeding pairs were also the fish sampled as adults. At the subadult (8 wpf; 50

- 51 dpf)

sampling, 8 of the 10 fish per aquaria were randomly sacrificed to a) produce the s ubadult samples and to

b) leave behind the breeding pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair (4

consecutive days per week over 5 weeks) in F0, F1, and F2; in adults in F0 , F1, and F2, length, weight,

VTG gene expression, SSC, and histopathology evaluation of gonad, kidney, and liver were recorded; and

in subadults in F1 and F2, VTG gene expression, SSC, gonad histology, weight and length were recorded.

In addition, hatch and survival from hatch to 3 wpf, 3 wpf to 8 wpf, and 9 wpf to 16 wpf were recorded.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with both the direction of the effect, and the

statistical test used indicated. The LOEC is co lor

- coded with more intense colors for lower

concentrations of trenbolone. In general, the F0 generation is less sensitive than the F1. Because there

were no viable fish available in the 32 and 84 ng/L treatments of F2, no effects were observed in any of

the measurements. In addition, the LOECs obtained at the F1 subadult sampling were lower than those ofthe F1 adult sampling. However, whether this was a result of a greater sensitivity at this lifestage or a

result of the statistical impact of greater sample size per replicate at the subadult sampling is difficult to

determine from the data. The LOEC for F1 fecundity/fertility was 32 ng/L, and the LOEC across the

entire trenbolone MMT ranged from 32 to 84 ng/L, predominately affecting XX fish.

The su ite of biological impacts on the fish within the MMT could be linked across levels of

biological organization to the well documented mode of action of trenbolone, as an AR agonist. At the

population/organismal level, reproduction, specifically fecundity, was reduced starting at 32 ng/L

presumably reflecting the affect trenbolone had on the phenotype of individual XX medaka. Given

intended usage of trenbolone is as an anabolic steroid to increase muscle mass in beef cattle, it was

somewhat surprising that a similar response in medaka, increased growth, was not observed consistently

during the course of the MMT. At the same time, a profound effect on the phenotype, the

individual/tissue level of organization, was observed in which individual XX fish develop ed into

phenotypic males starting at 32 ng/L. This was observed across levels of organization including

molecular (vitellogenin), tissue (external secondary sex characters and pathology), and organ (gonad

phenotype) all tethered back to the molecular init iating event: trenbolone binding and activating the AR.

Reproduction (Figure 1) :

In F0, both fecundity and fertility were reduced from the control level by the 84 ng/L exposure.

Interestingly, the reduction in fecundity and fertility was only statistically significant in the last week of

the assessment, all other weeks not different than controls, suggesting that trenbolone first reach ed

stea dy

- state in the tissue (or near to it) before an y activational affects would manifest as decreased

rep roductive output. In addition, the mean control fecundity was lower than would be hoped and

therefore there was also a lower mean number of fertile eggs as well, which resulted in both lower power

to discern effects and two statistically significant (but probably spurious) increases in treatment group

reproduction. Specifically, the 5 ng/L group had slightly increased fertility while the 2 ng/L group had

slightly increased fecundity.

In F1, fertility data was very similar to the fecundity data, indicati ng that any trenbolone

- induced

reproductive effects were the result of reduced fecundity. This generation also is a good example of the

challenges of the statistical analysis of relatively low power datasets. First off, the most obvious effect of

trenbol one exposure was at 32 and 84 ng/L, which eliminated all reproduction. However, the effects of

trenbolone at the other doses were less conclusive. For instance, 13 ng/L trenbolone seemed to reduce

fecundity if time was removed from the statistical model, however if time was included, the reduction was

only significant in the 3 rd and 5 th weeks of the assessment. Conversely, without time, both 2 and 5 ng/L

of trenbolone did not reduce reproduction; however both treatment groups had lower reproduction than

controls in one and two weeks, respectively. In short, trenbolone stopped reproduction at 32 ng/L and

above.

In F2, there were no affects on reproduction. However, this resulted from a lack of animals in the

two highest treatments: 32 and 84 ng/L t renbolone, and not any difference in sensitivity between the F1

and F2 generations.

Taken as a whole, trenbolone exposure did not affect fertility but did reduce fecundity at 32 ng/L

and above. While there was some indication that 13 ng/L trenbolone reduced fecundity in F1, it was not

borne out in the F2 generation. While this could have been because F1 was more sensitive than F2, it

seems more likely that these observations were a result of low replication within treatments and the

naturally high v ariance of reproduction. Lastly, the data strongly suggest that developmental impacts onreproduction are more sensitive than activational impacts as demonstrated by the lower LOEC in F1 than

in F0.

Adult Vitellogenin (Figure 2) :

Trenbolone had no effect on vitellogenin gene expression in F0 and F2 adults. Presumably the

XX fish in F0 were able to compensate for any potential androgenic effect of trenbolone , thu s no

reduction in vitellogenin gene expression was observed. In F2 there simply were no fish available for

sampling at both 32 and 84 ng/L so the only conclusion available wa s that the F2 LOEC was greater than

13 ng/L. There were effects observed in both XX and XY fish in F1. XY medaka had less vitellogenin

gene expression than the control XY fish when exposed to 32 and 84 ng/L trenbolone. While this was

certainly a statistically significant finding, it is difficult to ascribe a biological significance to a reduction

in male vitellogenin gene expression, and furthermore, this observation’s importance in defining an

EDC’s mode of action is not clear. Vitellogenin was also reduced in the XX fish exposed to 84 ng/L by

over 1500 times, in essence producing the male expression level. Trenbolone in other fish studies has

been shown to reduce vi tellogenin levels in females so this was expected from an AR agonist like

trenbolone.

Adult Secondary Sex Character s (Figure 3) :

There were no changes in the number of anal fin papillae in any of the XY fish across all three

generations. Also, there wa s no affect of trenbolone exposure observed on the XX fish in the F2

generation; however, this is clearly a result of not having any fish present in the 32 and 84 ng/L treatment

groups. On the other hand, there were increased papillae present in XX fish o f both F0 and F1 at 84 ng/L

trenbolone. In F0, there were also two XX fish in the 32 ng/L treatment with anal fin papillae. This

means that certainly 84 and probably 32 ng/L trenbolone was able to induce the development of papillae

over the 5 weeks of ex posure in F0 which started with XX adults . In F1, all XX fish in the 84 ng/L

treatment had papillae at levels equal to normal males, and trenbolone probably induced the development

of papillae in 2 XX individuals at 32 ng/L and 1 XX fish at 13 ng/L. Howe ver, there was also a single

XX medaka in the controls that had anal fin papillae at a level equal to a normal male. This individual

was tested multiple times with multiple methods for the presence of DMY and all tests came back

negative indicating with a strong likelihood that this individual medaka was a XX male. The

phenomenon of spontaneous XX males in medaka has been reported in the literature and observed in

culture at MED. These individuals are XX but are phenotypically normal males. Please note that this

individual was not removed from the analysis , including the statistics, because the effect of trenbolone is

identical to the “effect” seen in a XX male.

Adult Gonad phenotype (Figure 4) :

Again because of not having any fish in the two highest treatments (32 and 84 ng/L), there were

no effects on gonad phenotype observed in the F2 fish. Also, none of the XX fish in F0 had changes in

their gonad phenotypes, intersex or sex rever sal, which seemed to indicate that 5 weeks of exposure

starting with adults is not enough time to cause visible phenotypic changes in the gonad with an

androgen . In contrast, all the XX fish in the 84 ng/L treatment in F1 had testes. In addition there was a

single XX fish in the 13 ng/L treatment that also developed norm al testes. Considering there were no sex

reversed fish at this concentration in F2 adults nor was there any in the subadults of F1 and F2, this

individual is considered suspect and presumed to have been a spontaneous XX male as described

previously.

Adul t Pathology (Tables 2

– 3) :Only one pathology was observed at statistically significant levels in adult XY fish in F0, testicular

degeneration which was only observed at a severity score of 1 at 84 ng/L (Table 2) . On the other hand,

there were several m ore pathologies reported at significant levels in XX adults in this generation (Table

3) . In the kidney, hypereosinophilia was observed in the kidney at 32 ng/L with a severity of 2 and this

pathology increased in severity (3) at the higher concentration of 84 ng/L. There was also several

pathologies recorded in the gonads of these XX fish including mononuclear cell infiltration (severity = 1),

oocyte atresia (severity = 3), perifollicular cell hyperplasia/hypertrophy (severity = 2), “proteinaceous

fluid” (severity = 1), and decreased yolk formation (severity = 2). All the gonad pathologies were seen

only in the 84 ng/L trenbolone treatment group.

In F1 XY fish, there were two pathologies observed in the 84 ng/L treatment group (Table 4) . One,

asynchr onous development, was seen in the gonad at a severity of 1 while the other, tubule

mineralization, was observed in the kidney at a severity of 1. There were several pathologies observed in

the adult F1 XX fish (Table 5) , however these in general were not the same as the F0 fish and no

pathology was observed in the gonads. Specifically, the following were observed in the kidney at 84 ng/L

trenbolone: cellular debris in the tubule (severity = 1), and hypertrophy and hypereosinophilia of the

tubule epitheli um (severity = 3). In addition, minimal eosinophilia was seen in the liver at 32 ng/L and

above. Ironically, trenbolone at 84 ng/L so completely caused sex reversal in the XX individuals that the

pathologist observed no pathologies in the gonads of these animals, in spite of the fact that they were XX

fish with testes, albeit, histologically normal testes.

In F2, there was only two pathologies in adults (Table 6) , both in XX fish, and both were in the

kidney: hypertrophy and hypereosinophilia of the tubu le epithelium at a severity of 1 at 13 ng/L. There

were no pathologies in the XY fish of the F2 generation.

Adult Weight (Figure 5) :

The only statistically significant change in adult weight in the trenbolone MMT was in F0 XX fish

exposed to 84 ng/L in which there was an increase as compared to controls. There was a similar trend in

F1 XX fish at 32 ng/L that was not continued in the 84 ng/L treatment group presumably because those

XX fish were in fact

, phenotypic males and therefore weighed the same a s control males. Exposed XY

fish were the same as controls in all generations and treatments.

Adult Length (no figure) :

Trenbolone did not affect the length of either gender in any of the three generations.

Adult Survival (no figure) :

No effects were seen with trenbolone exposure to the survival of adult medaka from 7 through 15

wpf of either gender.

Subadult Vitellogenin (Figure 6) :

Trenbolone caused a decrease in vitellogenin gene expression at both 84 and 32 ng/L in subadult

XX medaka. At 32 ng/L, the vitellogenin mRNA copy number was lower by 2.6 times while at 84 ng/L

trenbolone, it was reduced by about 10,000 times. No effects were seen in XX, F2 fish presumably

because there were no fish at 32 ng/L and above. It did appear that the subadult LOEC was slightly less

than the adult LOEC within F1 (32 ng/L vs. 84 ng/L). However, this maybe a result of greater statistical

power due to higher sample sizes at the subadult life

- stage.There was no effect on XY fish in either F1 or F2, even though there seemed to be a similar trend

(reduction of vitellogenin mRNA) in both generations to that seen in the F1, XY adult samples. The

biological significance of these androgenic effects on XY fish is unclear.

Subadult Secondary Sex C haracters (Figure 7) :

Trenbolone induced the development of anal fin papillae in 9 out of 17 XX fish at 32 ng/L with

some of these fish having as many anal fin papillae as control males. After exposure to 84 ng/L, all the

subadult XX fish had the same number of anal fin papillae as the control males. As was the case for the

samples taken from adults of F2, there were no fish to be sampled in the 32 and 84 ng/L treatment groups

so no effects can be reported for F2.

Subadult Gonad Phenotype (Figure 8) :

As would have been expected given the data so far, especially the effect of trenbolone seen in the

F1 adults, XX subadult fish were all phenotypic males when exposed to 84 ng/L (16 out of 16 fish). On

the other hand, 7 of 10 XX fish exposed to 32 ng/L trenbolone were also phenotypic males which were

not observed at this concentration in the adult XX fish. However, there were only three XX fish that

survived the reproductive assessment making the adult sample size extremely small. In controls, there

w as a single XX male fish which we postulated to be a spontaneous occurrence. There wa s also an

intersex XX individual in the controls of F1. The pathologist’s observation was not of a gonad with both

ovarian and testicular tissue, but rather of a separate, distinct small testis within the adipose tissue of the

coelomic cavity. While included in the graph for complete reporting of the data, this is certainly an

individual that could be censored from the data as an outlier.

There were no changes f rom the expected gonad phenotype in F2. Again this would be expected

since there were no fish to be sampled in the 32 and 84 ng/L treatment groups.

Subadult Pathology (Tables 4

– 5) :

Across both generations, most of the pathologies reported as statisti cally significant were

observed at minimal severity (severity = 1) which is a subtle change from the normal state. Often

minimally severe findings are present even in controls, and always they are difficult to ascribe to a

biological consequence. For the sake of completeness, they have been included in this report; however,

they should be interpreted with caution.

In all the subadult XY fish from the entire trenbolone MMT, only two significant p athologies

were observed. In F1 (Table 7) there was mine ralization of the kidney tubule at 32 ng/L that appeared as

densely basophilic, granular material within the epithelium and/or lumens of various tubules. This

pathology was also observed in the F1 adult fish at 84 ng/L. The difference in life

- stage LOEC is

probably a result of lower power in the adults, on the other hand a LOEC was not observed in F2 because

the highest dose present was lower than this LOEC. In F2 (Table 9) , 4 of 20 fish had moderately severe

(severity = 4) dilation of the kidney tubule in the 13 ng/L treatment group. In these individuals,

approximately 80% of the tissue was affected giving the appearance of a polycystic kidney. This was the

most severe pathology observed in the entire MMT and one of the lowest LOECs seen for a single

p athology. However, it is unusual to have that marked response in only a single generation and life

- stage.

Kidney tubule dilation was not a significant observation in F2, even though the appropriate exposure

levels were present in F2, and it was not seen in adult XY fish in any of the generations.

The gonads of subadult XX medaka in F1 (Table 8) and F2 (Table 10) had only three significant

pathologies, apart from the obvious changes in gonad phenotype that have already been described. In F1only, ther e was decreased yolk at 32 ng/L indicated by pale eosinophilic yolk, absent yolk granules and

excessive vacuolation of the yolk. This was observed only at a minimally severe level. In both F1 and

F2, oocyte atresia was reported at very low concentration of 2 ng/L trenbolone. This pathology was

minimally (F2) or mildly severe (F1) and was observed as vacuolar hypertrophy of granulosa cells, loss of

yolk granules, and condensation of cortical alveolar material that at later stages of atresia progressed to

angular hypereosinophilic oocytes with coarsely granular cytoplasm and/or oocytes with absent

cytoplasm and collapsed, thickened vitelline envelopes. While oocyte atresia was observed at very low

concentrations of trenbolone, it was not very severe at any of the higher doses of trenbolone, presumably

because trenbolone

- effected animals were actually phenotypic males. Finally, proteinaceous fluid present

in the gonad was present in F2, subadult XX medaka only. It was always only minimally severe, and was

seen as free homogeneous material within the coelomic cavities of affected fish.

Pathologies of the kidney or liver of XX subadults were only found in the F1 generation. These

included kidney tubule epithelium hypereosinophilia and hypertrophy, kidney tubule epithelium

vacuolization, protein within kidney tubules, and liver eosinophilia. Kidney tubule epithelium

hypereosinophilia and hypertrophy were both present at a severity of 3 (moderate) in the 32 ng/L and

above treatment groups. The hypereosinophilia was characterized by a granular, hypereosinophilic,

cytoplasmic focus between the nucleus and basilar margin of tubular epithelial cells. This change in renal

tubular cytoplasm was often ac companied by hypertrophy of the cell and together concomitant with XX

male genotype/phenotype mismatch. Hypertrophy of the kidney tubule was was characterized by a 2

- 3x

increase in the size of renal tubular epithelial cells and a morphological change from sharply cuboidal

epithelium with striated eosinophilic cytoplasm to plump cuboidal to columnar epithelium with pale,

glassy eosinophilic cytoplasm and granular hypereosinophilic foci. Similarly and probably related to the

same pathological process in the kidney tubules, vacuolization and protein accumulation, were present

only at 84 ng/L and a severity of 1. Vacuolization was evident as clear, round to lacy vacuoles within the

cytoplasm of tubular epithelial cells while the protein was apparent as homoge nous eosinophilic material

within the lumens of renal tubules. In the liver, eosinophilia of hepatocytes was minimally observed in

these XX subadults at 32 and 84 ng/L and was characterized by a diffuse change in the livers of XX fish

from basophilic

- stai ning (hematoxylin) to eosinophilic staining (eosin).

Subdult Weight (Figure 9) :

There were no effects on weight in F2. In F1, XX fish in both the 32 and 84 ng/L treatment

groups weighed less than the controls. This was a result of the phenotypic cha nge from female to male,

induced in several on the XX fish exposed to 32 ng/L and all of the XX fish exposed to 84 ng/L of

trenbolone.

Subadult Length (no figure) :

Length was not affected by trenbolone exposure in any of the generations.

Subadult Sur vival (no figure) :

Unfortunately, the data was not collected to determine survival from hatch through 3 wpf.

Trenbolone did not lower survival from 4 wpf to 8 wpf at any of the tested concentrations in either

generation.

Subadult Hatch (no figure) :

Trenbolone had no affect on hatch in either the F1 or F2 generations at any of the tested

concentrations.Table 1. Summary table of LOECs for the various biological data collected during the MED trenbolone MMT.

LOECs are color co ded with more intense colors for lower concentrations. The direction of the change from controls is indicated by the directi on of the arrow

and statistical test used is abbreviated in the cell as well. JT=Jonckheere

- Terpstra, D=Dunnett, Du=Dunn Trenbolone MMT LOEC Summary Trenbolone

(ng/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

Subadult (8 wpf) Subadult (8 wpf) Adult (16 wpf) Adult (16 wpf) Adult (16 wpf) 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 2 0.5

Weight LOEC>84 Du:32 ↓ LOEC>13 LOEC>13 LOEC>84 D,JT:84 ↑ LOEC>84 LOEC>84 LOEC>13 LOEC>13 3 5 1.2

Length LOEC>84 LOEC>84 LOEC>13 LOEC>13 LOEC>84 LOEC>84 LOEC> 84 LOEC>84 LOEC>13 LOEC>13 4 13 2.7

Survival LOEC>84 LOEC>84 LOEC>13 LOEC>13 LOEC>84 LOEC>84 LOEC>84 LOEC>84 LOEC>13 LOEC>13 5 32 4.8

Hatch LOEC>84 LOEC>13 6 84 12.9

Papillae LOEC>84 Du,JT:32 ↑ LOEC>13 LOEC>13 LOEC>84 Du,JT:84 ↑ LOEC>84 Du,JT:84 ↑ LOEC>13 LOEC>13

Vtg LOEC>84 Du,JT:32 ↓ LOEC>13 LOEC>13 LOEC>84 LOEC>84 JT:32 ↓ D:84 ↓ LOEC>13 LOEC>13

Gonad

Phenotype LOEC>84 32 ↑ LOEC>13 LOEC>13 LOEC>84 LOEC>84 LOEC>84 84 ↑ LOEC>13 LOEC>13

Fecundity 84 ↓ 32 ↓ LOEC>13

Fertility 84 ↓ 32 ↓ LOEC>13

Highest Conc 84 13 84 84 13Figure 1, reproduction. Box plots of mean fecundity in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard

deviation, and the whisker is the standard error of the number of eggs

spawned per d ay by each replicate. Trenbolone F1 Reproduction MED MMT

0 2 5 13 32 84

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility Trenbolone F2 Reproduction MED MMT

0 2 5 13

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Trenbolone F0 Reproduction MED MMT

0 2 5 13 32 84

Concentration (ng/L) 0 5 10 15 20 25 30 35 40

Fecundity

FertilityFigure 2 , adult vitellogenin. Gender specific vitellogenin gene expression

of adult medaka in F1 (upper left), F2 (upper right), and F0 (lower left)

generations after exposed to trenbolone. The central line is the mean, the box

is the standard deviation and the whisker is the standard error of the

vitellogenin expression. Vitellogenin gene expression is graphed relative to

the gender specific controls. Significance is indicated by a letter: JT =

Jonckheere

- Terpstra and D = Dunnett’s. Trenbolone F2 Adult Vitellogenin MED MMT

Concentration (ng/L)Vtg mRNA Relative to Controls

0 2 5 13 32 84 0 2 4 6 8 10

XY

XXno viable F2 fishno viable F2 fish Trenbolone F1 Adult Vitellogenin MED MMT

Concentration (ng/L)Vtg mRNA Relative to Controls

0 2 5 13 32 84 0 2 4 6 8 10

XY

XX

D JT JT

Trenbolone F0 Adult Vitellogenin MED MMT

Concentration (ng/L)Vtg mRNA Relative to Controls

0 2 5 13 32 84 0 2 4 6 8 10

XY

XXFigure 3, adult secondary sex characters. Gender specific secondary sex

characters (SSC) of adult (16 wpf) medaka in F1 (upper left), F2 (upper

right), and F0 (lower left) generations after expos ure to trenbolone. The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the number of anal fin papillae, that is the SSC. Du or

JT = significantly different from controls as determined by Dunn’s test or

Jonckheere

- Terpstra trend test, respectively. Trenbolone F1 Adult SSC MED MMT

Concentration (ng/L)Anal Fin Papillae (#/fin)

0 2 5 13 32 84 0 20 40 60 80 100 120 140 160 180

Du,JT Trenbolone F2 Adult SSC MED MMT

Concentration (ng/L)Anal Fin Papillae (#/fin)

0 2 5 13 32 84 0 20 40 60 80 100 120 140 160 180

XY

XXno viable F2 fishno viable F2 fish

Trenbolone F0 Adult SSC MED MMT

Concentration (ng/L)Anal Fin Papillae (#/fin)

0 2 5 13 32 84 0 20 40 60 80 100 120 140 160 180

Du, JTFigure 4 , adult gonad phenotype. Gonad phenotype of XX adult (16 wpf)

medaka in F1 (upper left), F2 (upper right), and F0 (lower left) generations

after exposure to trenbolone. The stacked bar graphs show the percent of

individuals with testis, intersex, or ovary. The number in the boxes is the

number of individuals in each treatment with that gonad phenotype that is the

n. Intersex gonad is defined as a single gonad with any amoun t of both

ovarian and testicular tissue with no measurement of relative proportion. Sex

reversal is the presence of only testicular tissue in a XX fish. Trenbolone F1 Adult XX Gonad Phenotype MED MMT

Concentration (ng/L) . 0 2 5 13 32 84

. 0 20 40 60 80 100

5 5 6 6 3 1

3

intersex

tesits

ovary Trenbolone F0 Adult XX Gonad Phenotype MED MMT

Concentration (ng/L) . 0 2 5 13 32 84

. 0 20 40 60 80 100

6 6 6 6 6 6

intersex

tesits

ovary

Trenbolone F2 Adult XX Gonad Phenotype MED MMT

Concentration (ng/L) . 0 2 5 13 32 84

. 0 20 40 60 80 100

5 6 6 6

intersex

tesits

ovaryno fishno fish

### Table 2. Significant pathologies as indicated by RSCABS in adult XY medaka of the trenbolone MED MMT

F0 XY F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Testicular Degeneration 84 ng/L 1 84 ng/L 1

- - - - - - - - Asynchronous Develop

- - - - 84 ng/L 1 84 ng/L 1

- - - -

KIDNEY Tubule Mineralization

- - - - 84 ng/L 1 84 ng/L 1

- - - -



### Table 3. Significant pathologies as indicated by RSCABS in adult XX medaka of the trenbolone MED MMT

F0 XX F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc SeverityGONAD Cell Infiltrate, Mononuclear 84 ng/L 1 84 ng/L 1

- - - - - - - - Oocyte Atresia 84 ng/L 3 84 ng/L 3

- - - - - - - -

F ollicular Cell Hyperplasia Hypertrophy 84 ng/L 2 84 ng/L 2

- - - - - - - -

Proteinaceous Fluid 84 ng/L 1 84 ng/L 1

- - - - - - - -

Decreased Yolk Formation 84 ng/L 2 84 ng/L 2

- - - - - - - -

KIDNEY Tub Epith Eosinophilia 32 ng/L 2 84 ng/L 3 13 ng/L 1 84 ng/L 3 13 ng/L 1 13 ng/L 1

Tubule Debris Casts

- - - - 84 ng/L 1 84 ng/L 1

- - - -

Tub Epith Hypertrop hy

- - - - 13 ng/L 1 84 ng/L 3 13 ng/L 1 13 ng/L 1Figure 5, adult weight. Gender specific weights of adult (16 wpf) medaka

in F1 (upper left), F2 (upper right), and F0 (lower left) generations after

exposed to trenbolone. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of th e weight measured in mg.

D or JT = significantly different from controls as determined by Dunnett’s

test or Jonckheere

- Terpstra trend test, respectively. Trenbolone F0 Adult Weight MED MMT

Concentration (ng/L)Body Weight (mg)

0 2 5 13 32 84 0 200 400 600 800 1000 XY

XX

D,JT Trenbolone F2 Adult Weight MED MMT

Concentration (ng/L)Body Weight (mg)

0 2 5 13 32 84 0 200 400 600 800 1000

XY

XXno viable F2 fishno viable F2 fish Trenbolone F1 Adult Weight MED MMT

Concentration (ng/L)Body Weight (mg)

0 2 5 13 32 84 0 200 400 600 800 1000

XY

XXFigure 6, subadult vitellogenin. Gender specific vitellogenin gene

expression of suba dult (8 wpf) medaka in F1 (left), and F2 (right ) generations

after exposed to 4tOP. The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the vitellogenin expression.

Vitellogenin gene expression is graphed relative to the gender specific

controls. Du or JT = significantly different from controls as determined by

Dunn’s test or Jonckheere

- Terpstra

. Trenbolone F1 Subadult Vitellogenin MED MMT

Concentration (ng/L)Vtg mRNA Relative to Controls

0 2 5 13 32 84 0 2 4 6 8 10

XY

XX

Du,JT

Du,JT Trenbolone F2 Subadult Vitellogenin MED MMT

Concentration (ng/L)Vtg mRNA Relative to Controls

0 2 5 13 32 84 0 2 4 6 8 10

XY

XXno viable F2 fishno viable F2 fishFigure 7, subadult secondary sex characters. Gender specific secondary

sex characters (SSC) of sub adult (8 wpf) medaka in F1 (left), and F2 (right )

generations after exposure to trenbolone. The central line is the mean, the

box is the standard deviation and the whisker is the standard error of the

number of anal fin papillae that is the SSC. Du or JT = significantly

different from controls as determined by Dunn’s test or Jonckheere

- Terpstra

trend test, respectively. Trenbolone F2 Subadult SSC MED MMT

Concentration (ng/L)Anal Fin Papillae (#/fin)

0 2 5 13 32 84 0 20 40 60 80 100 120 140 160no viable F2 fishno viable F2 fish Trenbolone F1 Subadult SSC MED MMT

Concentration (ng/L)Anal Fin Papillae (#/fin)

0 2 5 13 32 84 0 20 40 60 80 100 120 140 160

Du,JT Du,JTFigure 8, subadult gonad phenotype. Gonad phenotype of XX subadult

medaka in F1 (left) and F2 (right ) after exposure to trenbolone. The stacked

bar graphs show the percent of individuals with testis, intersex, or ovary.

The number in the boxes is the nu mber of individuals in each treatment with

that gonad phenotype that is the n. Intersex gonad is defined as a single

gonad with any amount of both ovarian and testicular tissue with no

measurement of relative proportion. Trenbolone F2 Subadult XX Gonad Phenotype MED MMT

Concentration (ng/L) . 0 2 5 13 32 84

. 0 20 40 60 80 100

14no fishno fish 24 28 23 intersex

tesits

ovary Trenbolone F1 Subadult XX Gonad Phenotype MED MMT

Concentration (ng/L) . 0 2 5 13 32 84

. 0 20 40 60 80 100

25 25 16

10 7 27 1

1

19 intersex

tesits

ovaryTable 4

. Significant pathologies as indicated by RSCABS in subadult XY medaka of the trenbolone MED MMT

F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

KIDNEY Tubule Mineralization 32 ng/L 1 32 ng/L 1

- - - -

T u bule Dilation

- - - - 13 ng/L 4 13 ng/L 4

Table 5

. Significant pathologies as indicated by RSCABS in subadult XX medaka of the trenbolone MED MMT

F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Decreased Yolk 32 ng/L 1 32 ng/L 1

- - - - Oocyte Atresia 2 ng/L 2 2 ng/L 2 2 ng/L 1 2 ng/L 1

Proteinaceous Fluid

- - - - 13 ng/L 1 13 ng/L 1KIDNEY Tub Epith E osinophilia 32 ng/L 3 32 ng/L 3

- - - -

Tub Epith Vacuolization 84 ng/L 1 84 ng/L 1

- - - -

Tubules, Protein 84 ng/L 1 84 ng/L 1

- - - -

Tub Epith Hypertrophy 32 ng/L 3 32 ng/L 3

- - - -

LIVER Eosinophilia 32 ng/L 1 32 ng/L 1

- - - -Figure 9, subadult weight. Gender specific weight of suba dult (8 wpf)

medaka in F1 (right) and F2 (left ) generations after exposure to trenbolone.

The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the weight measured in mg. Du or JT =

significantly different from controls as determined by Dunn’s tes t or

Jonckheere

- Terpstra trend test, respectively. Trenbolone F1 Subadult Weight MED MMT

Concentration (ng/L)Body Weight (mg)

0 2 5 13 32 84 0 50 100 150 200 250 300 350 400

XY

XX Du Du Trenbolone F2 Subadult Weight MED MMT

Concentration (ng/L)Body Weight (mg)

0 2 5 13 32 84 0 50 100 150 200 250 300 350 400

XY

XXno viable F2 fishno viable F2 fishTest Chemical: prochloraz

MOA: aromatase inhibitor and probable AR antagonist

Protocol: full research MMT

Lab: MED

Summary of Test Chemical and MOA:

Prochloraz is a fu ngicide in which its efficacy is based about inhibition of sterol biosynthesis in

target organisms thereby limiting target organisms ability to synthesize cell walls. Because of the

conserved nature of the pathways involved, prochloraz also inhibits CYP19 aromatase in fish which

catalyzes the conversion of testosterone to 17β

- estradiol both in the ovary and the brain. In addition,

there is evidence that prochloraz acts as an AR antagonist. In the 21

- day fathead minnow reproductive

assay, prochloraz produ ces a suite of biological effects characteristic of an aromatase inhibitor and an AR

antagonist including alterations in various steroid levels, reduced vitellogenin in females, and lower

accumulative fecundity at 0.1 mg/L prochloraz.

Summary of Protocol:

The MMT completed by USEPA

- MED testing the effects of prochloraz was done with the full

research MMT protocol th at has been detailed previously. Briefly, the prochloraz MMT protocol spanned

2 1⁄2 generations, in which in the F0 generation chemical exposu re was initiated with spawning pairs at the

beginning of 11 wpf (76 dpf) and continues through the beginning of 16 wpf (111 dpf) at which time the

fish were sacrificed. In the F1 and F2 generations, fish were continuously exposed from embryo through

adult spawning. Fish were sampled at two major life

- stages; subadults (in the 8th wpf; 50

- 51 dpf) in the

F1 and F2 generations, and mature adults (at the start of the 16th wpf ; 111 dpf ) in F0, F1, and F2.

Reproduction was evaluated for 4 consecutive days per week over 5 weeks from 11 wpf to 16 wpf.

The general design employed 6 treatments including dilution water controls and 5 treatment

concentrations with 6 replicates for each treatment level for a total 36 replicate units (i.e. aquaria) . The

average mea sured prochloraz concentrations in the test were 5, 9, 17, 25, and 41 μ g/L. Reproduction in

all three generations (F0, F1, and F2) consisted, nominally, of 6 breeding pairs per concentration and 6

breeding pairs for controls. These breeding pairs were also the fish sampled as adults. At the subadult (8

wpf; 50

- 51 dpf) sampling , 8 of the 10 fish per aquaria were randomly sacrificed to a) produce the

subadult samples and by subtraction to b) leave behind the breeding pairs for each replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility f or each pair (4

consecutive days per week over 5 weeks) in F0, F1, and F2; in adults in F0, F1, and F2, len gth, weight

, SSC, and histopathology evaluation of gonad, kidney, and liver were recorded; and in subadults in F1 and

F2, VTG gene expression, SSC, g onad histology, weight and length were recorded. In addition, hatch

and survival from hatch to 3 wpf, 3 wpf to 8 wpf, and 9 wpf to 16 wpf were recorded.

Summary of Results (Table 1) :

The LOECs are summar ized in a “heat table” with both the direction of the effect . The LOEC is

color

- coded with more intense colors for lo wer concentrations of prochloraz

. In general, the F0

generation is less sensitive than the F1. In addition, the LOECs obtained at the subadult samplings were

generally lower than those of the adult samplings. However, whether this was a result of a greater

sensitivity at this lifestage or a result of the statistical impact of greater sample size per replicate at the

subadult sampling is difficult to determine from the data. The LOEC fo r fecundity was 41 μ g/L in F0, 25μ g/L in F2, and no LOEC was determined at the highest remaining concentration (25 μ g/L) in F1. The

LOECs across the entire prochloraz MMT ranged from 5 to 41 μ g/L, affecting both XY and XX fish.

Prochloraz reduced fecun dity at approximately 25 μ g/L, however the more profound effect was

the reduced hatch at 41 μ g/L in F1 that resulted in no individuals in that treatment for F1 reproduction.

Prochloraz also affected growth in relatively low concentrations but the decrease s observed in the

subadult samples were 1) not very large in absolute terms, and 2) were not present in the adults. In fact,

adults in F0 were actually larger than controls after exposure to prochloraz. In addition, there were

decreased copies of the vit ellogenin gene in XX fish, which can be plausibly linked to the inhibition of

aromatase, resulting in a decrease in circulating 17 β

- estrogen that in the end lowered hepatic vitellogenin

expression. Taken together, the MMT protocol was able to detect effec ts of prochloraz exposure across

the various levels of biological organization: individual/population level effects (hatch, growth, and

reproduction), tissue/organ level effects (pathology and SSC), and molecular (vitellogenin).

Reproduction (Figure 1 ) :

In only one week of F0, the 5 th week of the reproductive assessment, did any treatment have

significantly lower reproduction, both fecundity and fertility, than controls. Several of the treatments

were lower than controls in the 5 th week, however, controls seemed to be decreasing as well, suggesting a

general trend of ever decreasing reproductive output as the reproductive assessment progressed. The

mean number o f eggs spawned daily in the 41 μ g/L group was 17.3 in the first week, and thi s declined to

just 3 eggs per day with several of the pairs not producing any eggs in the 5 th week. In addition, the 41

μ g/L prochloraz group was determined to be statistically lower than controls in both fecundity and

fertility, not only in the 5 th week, but also by removing time from the model and analyzing via an

ANOVA with a Dunnett’s adjustment. There seemed to be a strong trend of decreasing reproduction

through time in F0, however

, because of the generally low statistical power of the MMT, it was n ot

detected by our statistical tests until the last week. By this time, the output of the pairs in the 41 μ g/L

treatment group was almost completely shutdown.

In F1, unfortunately the controls only had five breeding pairs because of a female mortality. In

addition, two of the remaining breeding pairs in the controls did not produce any eggs for the entire

assessment , a phenomenon that occurs occasionally. This greatly reduced the statistical power in an

already fairly low power study design to the exta nt that a mean fecundity of 4 .3 eggs per pair

- day in the

25 μ g/L treatment was not significantly lower than the controls (mean = 13.9 per pair

- day). While neither

was statistically significant, the data suggests an effect on f ecundity starting at 17 and 2 5 μ g/L prochloraz.

Since this MMT was completed, a power analysis has shown that the power to see a 30% reduction in

fecundity is only about 0.5 and to see a 40% reduction is still only 0.7, therefore it is not surprising to see

trends in the data that ar e not statistically significant.

In F2, all six available control breeding pairs spawned consistently high numbers of eggs

maximizing the statistical power of the reproductive assessment. F ecundity was reduced in the 25 μ g/L

treatment group in weeks 11

– 14

, while no other treatment, when using a repeated measures analysis,

was statistically lower than controls. It should be noted that while the reproductive output of the F2, 25

μ g/L treatment was statistically the same as the F1, 25 μ g/L treatment, a difference from controls was

observed in F2 and not in F1. Simply put, the control reproduction in F2 was much higher than the

controls of F1 leading to differing LOECs between the two generations. Given the lower power of this

MMT design , it is not surp rising to have significant differences from controls in one generation that was

not seen in another generation at the same concentration even though the general trends and character of

the data from the generations are quite similar.Adult Vitelloge nin (no data) :

Liver was not sampled in the adults so that a full histopathological analysis could be done that

retained the internal organs in vivo

.

Adult Secondary Sex Character s (Figure 2) :

Prochloraz did not alter the number of anal fin papillae in XY adults in any of the generations.

This is also true with the XX adults in all three generations. In F1, a single XX adult in each of the

controls, 5 μg/L, and 17 μg/L treatments had a high nu mber of papillae that was equivalent to what is

typically seen in normal adult males. These individuals are presumed to be spontaneous XX males which

have been detailed in the literature and have been observed at MED, and therefore are not a result of

exp osure to prochloraz.

Adult Gonad phenotype (Figure 3) :

Gonad phenotypes were simply one of three categories: testis, ovary, and intersex with any

number proportion of mixed phenotype gonad putting an individual in the intersex category. In all three

gene rations , prochloraz, at any of the con centrations tested, did not alter the gonad phenotype of either the

XX or XY fish. Again as noted previously, there were XX fish that were phenotypically male, in this

case had developed testes. Spontaneous XX ma les can arise in medaka which is what appears to have

happened in this MMT. There was also a single intersex XY fish in the F2, 5 μg/L treatment

, but this was

of very low severity and is likely not related to prochloraz exposure.

Adult Pathology (Tables 2

– 3) :

In adult XY medaka exposed to prochloraz in F0, F1, and F2, no pathology was observed with a

severity above that of a minimal finding (severity score = 1).

Adult XX fish in F0 had more pathologies with greater severity than what was seen in t he XY

fish of this generation. The moderately severe pathologies were oocyte dropout, post

- ovulatory follicles

and increased basophilia in the liver in the highest treatment (41 μg/L). Mildly severe pathologies that

were observed included decreased yolk formation in the 17 μg/L treatment group and above, increased

hepatocellular vacuolization in the 9 μg/L group, and gonadal follicular cell hyperplasia/hypertrophy in

the 41 μg/L treatment. Finally, minimally severe pathologies were that were observed inc luded cystic

degeneration in the liver after exposure to 41 μg/L.

In adult XX fish in F1, the only statistically significant pathology, a mild effect, was increased

quantity of post

- ovulatory follicles in the gonad.

In F2 adult XX fish, two pathologie s were seen at mild severity. Oocyte dropout in the gonad

was observed at 25 μg/L, and basophilia of the liver at a LOEC of 5 μg/L prochloraz.

Adult Weight (Figure 4) :

The weight in adult medaka, in either XX or XY fish, was not decreased by any prochl oraz

treatment in any of the generations. Weight was increased over controls in F0,XX fish in the 41 μg/L

prochloraz treatment group; controls had a mean weight of 567 mg while this treatment group had a meanof 680 mg, an increase of about 20%. However, no other effects were observed in adults and it is unclear

what negative biological impact an increase, even a 20% increase, would have on medaka.

Adult Length (Figure 5) :

The lengths of adult fish were affected by prochloraz positively in F0, negati vely in F1, and not at

all in F2. XX medaka in F0 were longer after exposure to 9, 17, 25, and 41 μg/L prochloraz than their

control counterparts. Conversely, XX and XY fish in F1 were shorter than controls in the 25 μg/L

treatment while no effect was se en in the other treatments tested. In addition, no difference was observed

in either XX or XY prochloraz exposed fish in F2 compared to controls. The difference in LOECs

between F0 and F1 may be explained by the obvious difference in exposure histories o f these two

generations, but this does not explain the difference between F1 and F2. However, as power analyses and

empirical data (i.e. experience) have suggested, the statistical power of the data collected in the adult

stage is unacceptably low, and mi ght have as much to do with the apparent intra

- generational differences

in sensitivity as actual differences

.

Adult Survival (Table 4) :

No effects were seen after prochloraz exposure to the survival of adult medaka from 8 to 16 wpf

of either gender.

Subadult Vitellogenin (Figure 6) :

In XY subadult fish from both F1 and F2 generations, prochloraz did not change the hepatic

expression level of the vitellogenin gene. However, prochloraz did lower the vitellogenin copy number in

livers from XX fish wit h a LOEC of 9 μg/L in both generations. In F1 controls

, the mean copy number

per ng of RNA was about 6 x 10 6 which decreased to about 1 x 10 6 , 0.4 x 10 6 , and 0.1 x 10 6 in the 9 μg/L,

17 μg/L, and 25 μg/L prochloraz treatment groups, respectively. A simi lar trend was apparent in the F2

treatments as well. The mean control copy number per ng of RNA was 4 x 10 6 which decreased to 1.7 x

10 6 and 0.4 x 10 6 in the 9 μg/L and 17 μg/L treatments, respectively.

Subadult Secondary Sex Characters (Figure 7) :

In F1 and F2, there were no effects on SSC by prochloraz on XX subadult fish at any

concentration tested. As a note, there were two presumptive XX males in the F1 subadult sampling.

In XY fish of F1, there was a slight decrease from controls in the number of anal fin papillae

starting at 9 μg/L. In the three concentrations where the decreased was observed, the absolute change was

fairly small; the control mean was 74 while the means for the 9 μg/L, 17 μg/L, and 25 μg/L treatments

were 35, 49, and 43, respe ctively. In addition these same three treatments had 2, 1, and 1 XY fish with no

papillae. In contrast, F2 had no statistically significant decrease in papillae, however, the means of the 9

μg/L and 17 μg/L treatments were similar to those measured in F1 : 49 and 32, respectively. In addition,

there were more cases of XY individuals in these two treatments with no papillae: 4 in the 9 μg/L

treatment and 7 in the 17 μg/L treatment. However, the control mean was much lower in F2 than F1, 55

versus 74, and there was single XY fish in F2 controls that had no papillae. Therefore, data in F2 was not

significantly different than controls even though similar data in F1 was.

It appears that prochloraz had a small effect on the development of SSC in subadult me daka with

a LOEC of 9 μg/L prochloraz (detected in F1) possibly by its action as an androgen antagonist. F1 and

F2 seemed to follow similar trends, however the lower control mean value observed in F2 seemed to limit

the ability to identify a LOEC in F2.Subadult Gonad Phenotype (Figure 8) :

There were no statistically significant changes in gonad phenotype observed in either F1 or F2 of

the prochloraz MMT. There were a few cases, including in controls, of minimal testicular oocytes

(intersex) in XY fish and four cases in the two generations of presumptive, spontaneous XX males.

Subadult Pathology (Tables 5

– 6) :

Several pathologies were observed with significant prevalence at minimal severity in subadults.

However, in F1 a single pathology, tubule dilatation in the liver, was observed at mild severity at 25 μg/L

in XY fish, and a single pathology in XX fish, gona d hypoplasiam was observed at mild severity at 25

μg/L. If F2, testicular oocytes were present with a mild prevalence at 17 μg/L in XY medaka, and post

ovulatory follicles in XX fish were found in the 17 μg/L treatment with severe severity.

Subdult Wei ght (Figure 9) :

Both F1 and F2 subadult fish had modestly decreased weight in both XX and XY fish. The

LOEC for XX and XY fish in F1 was 9 μg/L, and the LOEC for XX fish in F2 was also 9 μg/L.

However, the LOEC for XY fish in F2 was even lower at 5 μg/ L, the lowest concentration of prochloraz

tested in the MMT. Interestingly, the treatment groups higher than the LOEC did not seem to have a

more pronounced weight decrease. Also, the continuation of these effects into adulthood was not seen as

there wer e no effects on weight observed in the adult samples from F1 or F2.

Subadult Length (Figure 10) :

Because of increased sample sizes within replicates, the length measurement at the subadult stage

consistently detected decreased length at a LOEC of 9 μg/L for XX and XY fish in both F1 and F2, which

were not significant in the adult samplings. The decreases was fairly small in absolute terms in all cases.

For instance, in F1 control XX fish had a mean length of 27.0 mm while the most effected group (25

μg /L) had a mean length of 23.7, a decrease 12%. Also, the available groups higher than the LOECs had

about the same mean lengths so that it appeared that prochloraz, in the concentrations tested, decreased

length but only by the same amount regardless of c oncentration.

Subadult Survival (Table 7

- 8 ) :

Survival of subadults was measured in two different windows: early life stage (upon hatch to 4

wpf), and subadults (from 4 wpf to the subadult sampling at 8 wpf). There seemed to be reduced survival

of the early life stage fish with a LOEC of 25 μg/L in both F1 and F2, and also extremely low survival

(13%) in the 41 μg/L treatment. However, there did not ap pear to be decreased survival in the subadult

stage upon exposure to prochloraz.

Hatch (Table 9) :

An effect on hatch within the prochloraz MMT was seen at 25 μg/L in the F1 generation.

Decreased hatch in addition to lower survival in this treatment resulted in its loss from the F1, subadult

sampling onward.Table 1. Summary table of LOECs for the various biological data collected during the MED Prochloraz MMT.

LOECs are color coded with more intense colors for lower concentrations. The direction of the change from controls is indica ted by the direction of the arrow. Prochloraz MMT LOEC Summary Prochloraz ( μ g/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

subadult (8 wpf) subadult (8 wpf) adult (16 wpf) adult (16 wpf) adult (16 wpf) 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 5 1

Weight 9 ↓ 9 ↓ 5 ↓ 9 ↓ LOEC>41 41 ↑ LOEC>25 LOEC>25 LOEC>25 LOEC>25 3 9 2

Length 9 ↓ 9 ↓ 9 ↓ 9 ↓ LOEC>41 9 ↑ 25 ↓ 25 ↓ LOEC>25 LOEC>25 4 17 3

Survival LOEC>25 25 ↓ LOEC>41 LOEC>41 LOEC>25 LOEC>25 LOEC>25 LOEC>25 5 25 4

Hatch 25 ↓ LOEC>25 6 41 3

Papillae 9 ↓ LOEC>25 LOEC>25 LOEC>25 LOEC>41 LOEC>41 LOEC>25 LOEC>25 LOEC>25 LOEC>25

Vtg LOEC>25 9 ↓ LOEC>25 9 ↓

Gonad

Phenotype LOEC>25 LOEC>25 LOEC>25 LOEC>25 LOEC>41 LOEC>41 LOEC>25 LOEC>25 LOEC>25 LOEC>25

Fecundity 41 ↓ LOEC > 25 25 ↓

Fertility 41 ↓ LOEC > 25 25 ↓

Highest Conc 25/41 17/25 41 25 25Figure 1, reproduction. Box plots of mean fecundity and fertility in each

treatment in F1 (upper left), F2 (upper right), and F0 (lower left)

generations. The central line is the mean, the box is the standard deviation,

and the whisker is the standard error of the number of eggs spawned per

day by each replicate. Prochloraz F1 Reproduction MED MMT

0 5 9 17 25

Concentration (ug/L) 0 5 10 15 20 25 30 35 40 45

Fecundity

Fertility

Prochloraz F0 Reproduction MED MMT

0 5 9 17 25 41

Concentration (ug/L) 0 5 10 15 20 25 30 35 40 45

Fecundity

Fertility



# *

* Prochloraz F2 Reproduction MED MMT

0 5 9 17 25

Concentration (ug/L) 0 5 10 15 20 25 30 35 40 45

Fecundity

Fertility



# *

*Figure 2, adult secondary sex characters. Gender specific secondary sex

characters (SSC) of adult (16 wpf) medaka in F1 (upper left), F2 (upper

right), and F0 (lower left) ge nerations after exposure to prochloraz . The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the number of anal fin papillae, that is the SSC. D or JT

= significantly different from controls as determined by Dunnett’s test or

Jonckheere

- Terpstra trend test, respectively. Prochloraz-MED MMT F1 Adult SSC

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 5 9 17 25 0 20 40 60 80 100 120 140 160

XY

XX

Individual Fish

Prochloraz-MED MMT F0 Adult SSC

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 5 9 17 25 41 0 20 40 60 80 100 120 140 160

XY

XX

Individual Fish Prochloraz-MED MMT F2 Adult SSC

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 5 9 17 25 0 20 40 60 80 100 120 140 160

XY

XX

Individual FishFigure 3 , adult gonad phenotype. Gonad phenotype of XX and XY adult

(16 wpf) medaka in F1 (upper left), F2 (upper right), and F0 (lower left)

ge nerations after exposure to prochloraz . The stacked bar graphs show the

percent of individuals wi th testis, intersex, or ovary. Intersex gonad is

defined as a single gonad with any amount of both ovarian and testicular

tissue with no measurement of relative proportion

. Prochloraz-MED MMT F1 Adult Gonad Phenotype

Intersex

Testis

Ovary-0 XX5 XX9 XX17 XX25 XX41 XX0 XY5 XY9 XY17 XY25 XY41 XY- 0 20 40 60 80 100 Prochloraz-MED MMT F0 Adult Gonad Phenotype

Intersex

Testis

Ovary-0 XX5 XX9 XX17 XX25 XX41 XX0 XY5 XY9 XY17 XY25 XY41 XY- 0 20 40 60 80 100

Prochloraz-MED MMT F2 Adult Gonad Phenotype

Intersex

Testis

Ovary-0 XX5 XX9 XX17 XX25 XX41 XX0 XY5 XY9 XY17 XY25 XY41 XY- 0 20 40 60 80 100

### Table 2. Significant pathologies as indicated by RSCABS in adult XY medaka of the Prochloraz MED MMT

F0 XY F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Intraluminal histiocytes 345 μ g/L 1 345 μ g/L 1

- - - - - - - - Asynchronous

development/malformation - - - - 25 μg/L 1 25 μg/L 1

- - - -

Interstitial cell

hyperplasia/hypertrophy - - - - 25 μg/L 1 25 μg/L 1 9 μ g/L 1 9 μ g/L 1

KIDNEY Cellular debris, tubules 9 μ g/L 1 9 μ g/L 1

- - - - - - - -

Regenerative hyperplasia 25 μ g/L 1 25 μ g/L 1

- - - - - - - -

Tubule mineralization

- - - - 17 μg/L 1 17 μg/L 1 25 μ g/L 1 25 μ g/L 1

LIVER Cystic degeneration 41 μ g/L 1 41 μ g/L 1

- - - - - - - -



### Table 3. Significant pathologies as indicated by RSCABS in adult XX medaka of the Prochloraz MED MMT

F0 XX F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Decreased yolk 9 μg/L 1 17 μ g/L 2

- - - - 9 μg/L 1 9 μg/L 1

Follicular

hyperplasia/hypertrophy 9 μg/L 1 41 μ g/L 2

- - - - 17 μ g/L 1 17 μ g/L 1

Post

- ovulatory follicles 9 μg/L 2 41 μ g/L 3 5 μ g/L 1 25 μ g/L 2

- - - - Oocyte dropout 41 μg/L 3 41 μ g/L 3

- - - - 25 μ g/L 2 25 μ g/L 2LIVER Basophilia 41 μg/L 3 41 μ g/L 3

- - - - 5 μ g/L 2 5 μ g/L 2

Cystic degeneration 41 μ g/L 1 41 μ g/L 1

- - - - - - - -

Increased vacuolization 5 μ g/L 1 9 μg/L 2

- - - - - - - -

Granulomatous inflammation

- - - - - - - - 25 μg/L 1 25 μg/L 1

Peribiliary fibrosis

- - - - - - - - 25 μg/L 1 25 μg/L 1Figure 4 , adult weight. Gender specific weights of adult (15 wpf) medaka

in F1 (upper left), F2 (upper right), and F0 (lower left) g enerations after

exposed to prochloraz . The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the weight measured in mg.

JT = significantly different from controls as determined by Jonckheere

Terpstra trend test. Prochloraz-MED MMT F1 Adult Weight

Concentration (ug/L)Body Weight (mg)

0 5 9 17 25 0 100 200 300 400 500 600 700 800 900 1000

XY

XX

Prochloraz-MED MMT F0 Adult Weight

Concentration (ug/L)Body Weight (mg)

0 5 9 17 25 41 0 200 400 600 800 1000 1200

XY

XX

JT Prochloraz-MED MMT F2 Adult Weight

Concentration (ug/L)Body Weight (mg)

0 5 9 17 25 0 200 400 600 800 1000 1200 1400

XY

XXFigure 5, adult length. Gend er specific lengths of adult (16 wpf) medaka in

F1 (upper left), F2 (upper right), and F0 (lower left) generations after

exposed to prochloraz . The central line is the mean, the box is the standard

deviation and the wh isker is the standard error of the weight measured in mg.

JT = significantly different from controls as determined b y Jonckheere

Terpstra trend. Prochloraz-MED MMT F1 Adult Length

Concentration (ug/L)Length (mm)

0 5 9 17 25 0 30 32 34 36 38 40 42 44 46 48

XY

XX JT JT Prochloraz-MED MMT F2 Adult Length

Concentration (ug/L)Length (mm)

0 5 9 17 25 0 32 34 36 38 40 42 44

XX

XY

Prochloraz-MED MMT F0 Adult Length

ConcentrationLength (mm)

0 5 9 17 25 41 0 34 36 38 40 42

XY

XX

JT JT JT

JTTable 4. Survival from 8 wpf to 16 wpf.

Adult Survival (mean %, (N))

Treatment

Generation control 5 μg/L 9 μg/L 17 μg/L 25 μg/L 41 μg/L

F0 100 (6) 100 (6) 100 (6) 100 (6) 100 (6) 100 (6)

F1 100 (6) 100 (6) 100 (6) 100 (6) 66 (6)

F2 100 (6) 100 (6) 100 (6) 100 (6) 83 (6)Figure 6, subadult vitellogenin. Gender specific vitellogenin gene

expression of subadult medaka in F1 (left), and F2 (right) generations. The

central line is the mean, the box is the standard deviation and the whisker is

the standard error of the vitellogenin expression. Significance is indicated by

D = Dunnett’s

. Prochloraz-MED MMT F1 Subadult Vitellogenin

Concentration (ug/L)Vitellogenin mRNA Copies/ng RNA

0 5 9 17 25 41 0 2000000 4000000 6000000 8000000 10000000

XY

XX

D

D D Prochloraz-MED MMT F2 Subadult Vitellogenin

Concentration (ug/L)Vitellogenin mRNA Copies/ng RNA

0 5 9 17 25 41 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000

XY

XX

D

DFigure 7, subadult secondary sex characters. Gender specific secondary

sex characters (SSC) of sub adult (8 wpf) medaka in F1 (left), and F2 (right )

generations after exposure to prochloraz . The central line is the mean, the

box is the standard deviation and the whisker is the standard error of the

number of anal fin papillae that is the SSC. JT = significantly different

from controls as determined by Jonckheere

- T erpstra trend test. Prochloraz-MED MMT F1 Subadult SSC

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 5 9 17 25 0 20 40 60 80 100 120 140

XY

XX

Individual Fish

JT JT

JT Prochloraz-MED MMT F2 Subadult SSC

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 5 9 17 0 20 40 60 80 100 XY

XX

Individual FishFigure 8, subadult gonad phenotype. Gonad phenotype of subadult

medaka in F1 (left) and F2 (right) after exposure to prochloraz . The stacked

bar graphs show the percent of individuals with testis, intersex, or ovary.

The number is the number of individu als in each treatment with that gonad

phenotype that is the n. Intersex gonad is defined as a single gonad with any

amount of both ovarian and testicular tissue with no measurement of relative

proportion. Prochloraz-MED MMT F1 Subadult Gonad Phenotype

Intersex

Testis

Ovary-0 XX5 XX9 XX17 XX25 XX41 XX0 XY5 XY9 XY17 XY25 XY41 XY- 0 20 40 60 80 100

17 15 25 21 13 16 13 1

1 1

5 1

26 30 Prochloraz-MED MMT F2 Subadult Gonad Phenotype

Intersex

Testis

Ovary-0 XX5 XX9 XX17 XX25 XX41 XX0 XY5 XY9 XY17 XY25 XY41 XY- 0 20 40 60 80 100

24 9 2 2 3 1

2

23 13 17 25 19 18Table 5

. Significant pathologies as indicated by RSCABS in subadult XY medaka of the Prochloraz MED MMT

F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Interstitial cell

hyperplasia/hypertrophy 9 μg/L 1 9 μg/L 1 9 μg/L 1 9 μg/L 1

Decreased spermatozoa 17 μg/L 1 17 μg/L 1 17 μg/L 1 17 μg/L 1

Degeneration 9 μg/L 1 9 μg/L 1

- - - -

KIDNEY Dilation 25 μg/L 2 25 μg/L 2 17 μg/L 1 17 μg/L 1

Cellular debris, tubules

- - - - 17 μg/L 1 17 μg/L 1

Table 6

. Significant pathologies as indicated by RSCABS in subadult XX medaka of the Prochloraz MED MMT

F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Hypoplasia 25 μg/L 2 25 μg/L 2

- - - -

Post

- ovulatory follicles 9 μg/L 1 9 μg/L 1 17 μg/L 4 17 μg/L 4

Oocyte atresia

- - - - 17 μg/L 1 17 μg/L 1

Follicular

hyperplasia/hypertrophy - - - - 9 μg/L 1 9 μg/L 1Figure 9, subadult weight. Gender specific weight of sub adult (8 wpf)

medaka in F1 (left), and F2 (right ) generations after exposure to prochloraz

. The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the weight. JT or D = significantly different

from controls as determined by Jonckheere

- T erpstra trend test or Dunnett

test

. Prochloraz-MED MMT F1 Subadult Weight

Concentration (ug/L)Body Weight (mg)

0 5 9 17 25 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

XY

XX D

JT JT JT D D Prochloraz-MED MMT F2 Subadult Weight

Concentration (ug/L)Body Weight (ug/L)

0 5 9 17 0 50 100 150 200 250 300 350 400

XY

XX

D

D D

D DFigure 10, subadult length. Gender specific length of sub adult (8 wpf)

medaka in F1 (left), and F2 (right ) generations after exposure to prochloraz

. The central line is the mean, the box is the standard deviation and the

whisker is the standard error of the length (mm)

. Prochloraz-MED MMT F1 Subadult Length

Concentration (ug/L)Length (mm)

0 5 9 17 25 0 5 10 15 20 25 30 35

XY

XX D D D

JT JT JT Prochloraz-MED MMT F2 Subadult Length

Concentration (ug/L)Length (mm)

0 5 9 17 0 5 10 15 20 25 30 35 40

XX

XY JT JT JT

JTTable 7. Early life survival (hatch to 4 wpf).

Survival (mean %, (N))

Treatment

Generation control 5 μg/L 9 μg/L 17 μg/L 25 μg/L 41 μg/L

F1 76 63 69 52 34 13

F2 76 73 68 100 45

-

Table 8. Subadult survival (4 wpf to 8 wpf)

Survival (mean % )

Treatment

Generation control 5 μg/L 9 μg/L 17 μg/L 25 μg/L 41 μg/L

F1 95 95 88 85 94

-

F2 97 98 93 95 71

-

Table 9. Hatch

Hatch (mean % )

Treatment

Generation control 5 μg/L 9 μg/L 17 μg/L 25 μg/L 41 μg/L

F1 85 91 96 86 67 27

F2 94 82 92 87 91

-Test Chemical: vinclozolin

MOA: anti

- androgen

Protocol: abbreviated MMT

Lab: MED

Summary of Test Chemical and MOA:

Vinclozolin and its metabolites (M1 and M2) are anti

- androgens that via primarily through the metabolites also

persist in the environment (half life of M1 and M2 is over 180 days). Vinclozolin is a dicarbozimide fungicide that is

commonly used to fruits, vegetables, ornamental plants, and turf grasses, and therefore is an enviromentally relevant EDC.

THE MOA of vinclozolin is to bind with the androgen receptor, but not initiate the transcription of androgen

- dependent

genes.

Summary of Protocol:

The MMT completed by USEPA

- MED testing the effects of vinclozolin was done with an abbreviated MMT

protocol. Specifically, vinclozolin exposure was initiated in F0 in 12 w pf (83 dpf) and continued in F0 until the 16 wpf

(113 dpf) at which time the fish were sacrificed. From 90

- 110 dpf of F0, reproduction was monitored. In the F1

generation , fish were continuously exposed from embryo through adult spawning. Fish were sam pled at two major life

stages; subadults (in the 9th wpf; 59

- 60 dpf), and mature adults ( in the 15th wpf ; 101 dpf). F1 r eproduction was

evaluated for 3 week s from 12 wpf to 14 wpf (77 dpf

– 97 dpf)

. In the F2 generation , fish were continuously exposed

f ro m embryo through subadult at which time they were sacrificed (in the 9th wpf; 59

- 60 dpf).

The general design employed 6 treatments including dilution water controls and 5 treatment concentrations with 6

replicates for each treatment level for a total 36 replicate units (i.e. aquaria). The proportionate chemical concentration

difference between treatments was 0.5. The average measured vinclozolin concentrations in the test were 17, 33, 70, 136,

and 253 μ g/L. Reproduction in F0 and F1 consisted, nominally, of 6 breeding pairs per concentration and 6 breeding pairs

for controls. These breeding pairs were also the fish sampled as adults. At the subadult sampling, 10 of the 12 fish per

aquaria were randomly sa crificed to a) produce the subadult samples and to b) leave behind the breeding pairs for each

replicate unit.

The biological data obtained in this MMT included daily fecundity and fertility for each pair in F0 and F1; in

adults in F0 and F1, length, wei ght, VTG gene expression, SSC, and histopathology evaluation of gonad, kidney, and liver

were recorded (F1 only); and in subadults in F1 and F2, VTG gene expression, SSC, gonad histology, weight and length

were recorded. In addition, hatch and survival fr om hatch to 3 wpf, 3 wpf to 9 wpf, and 9 wpf to 14 wpf were recorded.

Summary of Results (Table 1):

The LOECs are summarized in a “heat table” with both the direction of the effect, and the statistical test used

indicated. The LOEC is color

- coded with more intense colors for lower concentrations of 4tOP. There were no viable

XY fish available in the 253 μ g/L treatment of F1 and both the 136 and 253 μ g/L treatments of F2. The LOEC for

fecundity and fertility, arguably the most important endpoints, was 17 μ g/L in F1, but no effects on reproduction were

observed in F1. The LOECs across the entire vinclozolin MMT ranged from 17 to 253 μ g/L.

Reproduction (Figure 1):

In F0, fecundity decreased in the 253 μ g/L treatment starting in the second week of the reproduction assessment

which is the third week after the start of exposure to vinclozolin. The mean for the entire three week reproduction

assessemtn was just 7 eggs spawned per day as compared to 24 spawned eggs per day in controls. Statistically sign ificant

reduced numbers of spawned eggs were also seen in all the other treatment groups (17, 33, 70, and 136 μ g/L ) in the last

week of the assessment, but this reduction was not great in absolute terms: controls at 24 eggs per day and all the

treatments a t approximately 15 spawned eggs per day.In F1, there was no effects of vinclozolin exposure at the concentrations tested. The mean fecundities observed

were 25, 26, 20, 29 (only 1 pair), and 26 in the controls, 17, 33, 70, and 136 μ g/L , respectively. In addition, there was

almost no reproduction in the 253 μ g/L treatment, however there was only a single breeding pair in this treatment.

The percentage of fertile eggs was ?????

Adult Vitellogenin (no figure) :

Livers remained in situ for histopathology, therefore no vitellogenin analysis was done in adult fish.

Adult Secondary Sex Characters (Figure 2):

Vinclozolin exposure had no effect on SSC in adult of either F0 or F1 generations. There were three XY fish in

the control of F1 that did not have any anal fin papillae. This is an extremely unusual and unexplained result especially in

light of observed normal fecundity/fertility in the same control fish. Any explanation is speculative, but it seems likely

that the zero pap illae counts in the control XY fish were erroneous.

Adult Gonad phenotype (no figure ):

Adults in only F1 were analyzed histologically. No alterations of gonad phenotype were observed in both XX and

XY fish at all levels of exposure.

Adult Pathology (Tables 2

– 3):

Multiple organs including gonad, liver, and kidney were analyzed by the pathologist for lesions resulting from

exposure to vinclozolin. The only generation in which potential pathologies were assessed was F1. In the XY fish, t he

only statistically significant pathologies were found in the gonad of vinclozolin exposed fish. Specifically, minimal

testicular degeneration (severity = 1) and mild interstitial cell hypertrophy (severity = 2) was noted in the highest dose

with XY fis h available, 136 μ g/L vinclozolin. In addition, minimal mineralization (severity = 1) in the testes was

observed in all the treatments (17 μ g/L through 136 μ g/L ).

In the XX fish of F1, only a single pathology was found to be statistically significant: interrenal

- chromaffin cell

hyperplasia was evident at a minimal severity in both the 136 and 253 μ g/L treatments. However, the severity did not

increase concurrently with increased concentration of vinclozolin.

Adult Weight (Figure 3):

Across both F0 and F1, vinclozolin exposure, in general, resulted in a slight increase in body weight. Specifically

in F0, the XX LOEC was 253 μ g/L in which the mean weight increased from 381 mg in control fish to 518 mg. The

LOEC in F0, XY fish was the 33 μ g/L treatment in which the mean weight increased from 275 mg in control fish to 337

in this treatment. Similar mean body weights were also observed in higher treatments with means at 70 μ g/L , 136 μ g/L

, and 253 μ g/L vinclozolin of 336 mg, 322 mg, and 3 61 mg, respectively.

Interestingly, increased body weight was not observed in adult F1 XY fish at any of the concentrations tested

(highest treatment with XY fish was 136 μ g/L ). On the other hand, XX fish in F1 weighed significantly more than

controls starting with those in the 70 μ g/L treatment (313 mg vs. 477 mg) and continuing with the higher treatments of

136 μ g/L and 253 μ g/L vinclozolin. In these two highest concentrations, the mean body weights were 439 mg and 493

mg, respectively, as compared to 313 mg in control XX fish.

Adult Length (Figure 4):

Similar to the effects on weight, vinclozolin, when an affect was observed, increased the length of fish in both F0

and F1. In F0, the only effect was slightly increased length in the XY fish of t he 253 μ g/L treatment from a mean of 32.5mm in control versus 34.3 in this highest treatment group. In F1, length was not altered in XY fish at any of the

concentrations of vinclozolin. On the other hand, vinclozolin exposure significantly increased the length of XX adult fish

in F1 starting at 70 μ g/L and continuing to 136 μ g/L (there was only a single XX fish in the 253 μ g/L group) by about

10% over controls (32.2 mm in the control, 35.5 mm in the 70 μ g/L treatment, and 36.1 mm in the 136 μ g/L treatmen t).

Adult Survival (no table):

No effects were seen after vinclozolin exposure to the survival of adult medaka from 9 to 15 wpf of either gender.

Subadult Vitellogenin (Figure 5):

No effects on vitellogenin gene expression were seen in either XX or XY fish from the F0 generation. There were

extremely high values (1520 and 3390 times that of the mean control) in 2 of the XY fish of the 33 μ g/L treatment group.

Not only are these mu ch high than the typical values recorded for control XY fish, they are equal to or higher than the

copies per ng RNA found in the control XX fish.

XY fish in the 136 μ g/L treatment, the highest concentration remaining in F2, had elevated expression of

vitellogenin, almost 24X higher copy number than their control counterparts. However, the elevated mean for the 136

μ g/L treatment was largely driven by three extreme values of 154X, 246X, and 404X of the mean control value of XY

fish. Statistically thes e values are outliers, but there is no biological or systematic (i.e. human error) reason to exclude the

data, therefore both they and the outliers seen in F0 were kept in the dataset for analysis.

Subadult Secondary Sex Characters (Figure 6):

In F1 subadult, XY fish exposed to vinclozolin, the mean number of anal fin papillae was lower than controls in

both 17 and 33 μ g/L treatments. Unfortunately, there were no males available in the 70 μ g/L treatment group, and only a

single XY fish in the 136 μ g /L treatment with 27 anal fin papillae (much lower than the control mean of 78). While the

mean number of papillae in XY fish for each of the treatments was lower in F2 than the corresponding treatment in F1,

statistically only the 33 and 136 μ g/L treatm ents were lower than the F2 XY control. This was because the XY controls in

F2 were much lower than in F1 (55 in F2 versus 78 in F1). The general trend seemed to be decreased SSC at relatively

low vinclozolin concentrations.

There were no effects on SS C in either generation’s XX fish. The only observation of note was the high number

of presumptive XX males interspersed throughout the treatments in both generations.

Subadult Gonad Phenotype (no figure ):

Vinclozolin did not appear to alter the gonad phenotype of XX or XY fish in F1 or F2 generation. However, there

were four F1, XY fish in the 33 μ g/L treatment (the highest treatment with XY fish available for histology) that had

normal ovaries, no anal fi n papillae, and high vitellogenin levels (equal to normal XX levels). In addition, there were three

F2, XY fish in the 136 μ g/L treatment (again the highest treatment with XY fish available for histology) also had the same

phenotypes: normal ovaries, no an al fin papillae, and high vitellogenin levels. There are two explanations for these

observations. First, contamination into XX samples from XY fish during the fin clip/DMY assessment procedure would

result in this very pattern. Second, the data could be appropriate and simply reflected a biological response to vinclozolin

exposure. However, this seems unlikely because certain aspects of the complete dataset do not seem to follow the expect

pattern from an EDC with that strong of an affect on the gonad. For instance, there were no instances of XY fish with

intersex gonads in the effected treatments or the lower treatments. There was not the suite of pathologies in the gonad or

liver that would be easily related to an EDC exposure.

While our interp retation of the data points to contamination, it is impossible to make a definitive statement

without a re

- assessment of DMY. This can be done on the fixed, paraffin embedded tissue remaining after histology, but

we have been unable to successful extract DNA of sufficient quality to perform the DMY assessment on these samples.

We suspect the use of Bouin’s fixative, specifically the picric acid component, is the primary issue.Subadult Pathology (Tables 4

– 5 ):

While the RSCABS statistical algorithm f ound a few significant pathologies, often these were not deemed

significant by an experienced pathologist. Conversely some of the observations deemed significant by the pathologist

were not statistically significant. The summary provided by the pathologi sts for XY fish included

“The predominant effect of vinclozolin treatment in male fish was interstitial cell hyperplasia

- hypertrophy. This

was observed as a treatment

- related finding sub

- adult males of the F 2 generation, but not in sub

- adult males of the F 1 generation. However, the overall prevalence of interstitial cell hyperplasia

- hypertrophy in F 2 sub

- adults was not high,

and the severity of the finding was never greater than minimal to mild. ”

The summary provided by the pathologists for XX fish included

“The principal effects of vinclozolin exposure in female medaka were increased ovarian lobe separation in sub

adults of both the F 1 and F 2 generations, and an elevated severity of increased ovarian oocyte atresia in F 1 adults.

Although, increased oocyte atresia is a common response to endocrine disruptors, systemic toxicity, and/or stress,

increased ovarian lobe separation is a novel finding for which the causative mechanism is undetermined.”

Subdult Wei ght (Figure 7 ):

There were no effects on weight in the subadults in either F1 or F2 generations.

Subadult Length (Figure 8 ):

There were no effects on length in the subadults in either F1 or F2 generations.

Subadult Survival (Table 6

- 7):

Vinclozolin decreased survival of embryos from hatch to 3 wpf at the highest treatment tested: 253 μg/L from a

mean survival of 84% in controls to only 25% in this treatment in F1. (Note: there was no 253 μg/L treatment in F2.)

Survival from 3 wpf to 9 w pf was the same in all the other treatments and controls in both F1 and F2; in fact there was

only a small number of deaths across all treatments in both generations. The mean survival in any of the treatments across

the MMT was 99

- 100% between 3 wpf and 9 wpf.

Subadult Hatch (Table 8 ):

The only effect seen in the vinclozolin MMT on hatch was in the 253 μg/L treatment of the F1 generation. This

treatment had a hatch of 65% as compared to 97% in the controls of F1 generation. This treatment group also had low

survival from hatch to 3 wpf.Vinclozolin MMT MED LOEC Summary Vinclolozlin ( μ g/L)

F1 F2 F0 F1 F2 Trt Conc S.D.

Subadult (9 wpf) Subadult (9 wpf) Adult (15 wpf) Adult (15 wpf) Adult 1 0 0

XY XX XY XX XY XX XY XX XY XX 2 17 2

Weight LOEC>136 LOEC>253 LOEC>136 LOEC>136 33 ↑ (JT) 253 ↑ (JT) LOEC>136 70 ↑ (JT) 3 33 3

Length LOEC>136 LOEC>253 LOEC>136 LOEC>136 253 ↑ (JT) LOEC>253 LOEC>136 70 ↑ (JT) 4 70 6

Survival 253 ↓ LOEC>136 LOEC>253 LOEC>253 LOEC>136 LOEC>253 5 136 18

Hatch 253 ↓ need stats LOEC>136 6 253 59

Papillae 17 ↓ (JT) LOEC>253 33 ↓ (JT) LOEC>136 LOEC>253 LOEC>253 LOEC>136 LOEC>253

Vtg LOEC>136 LOEC>253 136↑ (JT) LOEC>136

Gonad

Phenotype LOEC>136 LOEC>253 LOEC>136 LOEC>136 LOEC>136 LOEC>253

Fecundity 17 ↓ (in 3rd week) LOEC>136

Fertility LOEC>253(=fecundity) LOEC>136

Highest

Conc 136/253 136 253 136/253Figure 1, reproduction. Box plots of mean fecundity and fertility in each treatment in F0 (upper) and F1 (lower)

generations. The central line is the mean, the box is the standard deviation, and the whisker is the standard error of the

number of eggs spawned or the number of fer tile eggs spawned per day by each replicate. Vinclozolin F0 Reproduction MED MMT

0 17 33 70 136 253

Concentration (ug/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Raw Data

Vinclozolin F1 Reproduction MED MMT

0 17 33 70 138 253

Concentration (ug/L) 0 5 10 15 20 25 30 35 40

Fecundity

Fertility

Raw DataFigure 2, adult secondary sex characters. Gender specific secondary sex characters (SSC) of adult (15 wpf) medaka in F0

(lower) and F1 (upper) generations after exposure to vinclozolin. The central li ne is the mean, the box is the standard

deviation and the whisker is the standard error of the number of anal fin papillae, that is the SSC. Vinclozolin F0 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 17 33 70 136 253 0 20 40 60 80 100 120 140 160 180

XY

XX

Vinclozolin F1 Adult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 17 33 70 136 253 0 20 40 60 80 100 120 140 160 180

XY

XX

### Table 2. Significant pathologies as indicated by RSCABS in adult XY medaka of the Vinclozolin MED MMT

Table 3. Significant pathologies as indicated by RSCABS in adult XX medaka of the Vinclozolin MED MMT F1 X Y

LOEC Highest Severity

Conc Severity Conc Severity

Gonad Testicular Degeneration 136 μg/L 1 136 μg/L 1

Mineralization 17 μ g/L 1 17 μg/L 1

Interstitial Cell Hypertrophy 136 μg/L 2 136 μg/L 2

F1 XX

LOEC Highest Severity

Conc Severity Conc Severity

Kidney Interrenal

- Chromaffin Cell

Hyperplasia 136 μg/L 2 136 μg/L 2Figure 3, adult weight. Gender specific weights of adult (15 wpf) medaka in F0 (upper) and F1 (lower) generations after

exposure to vinclozolin. The central line is the mean, the box is the standard deviation and the whisker is the standard

error of the weight measured in mg. J T = significantly different from controls as determined by Jonckheere

- Terpstra

trend test. Vinclozolin F0 Adult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 17 33 70 136 253 0 100 200 300 400 500 600 700 800

XY

XX

JT JT

JT

JT JT

Vinclozolin F1 Adult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 17 33 70 136 253 0 100 200 300 400 500 600 700 800

XY

XX

JT JT

JTFigure 4, adult length. Gender specific lengths of adult (15 wpf) medaka in F0 (upper) and F1 (lower) generations after

exposure to vinclozolin. The cent ral line is the mean, the box is the standard deviation and the whisker is the standard

error of the weight measured in mg. JT = significantly different from controls as determined by Jonckheere

- Terpstra

trend test. Vinclozolin F0 Adult Length MED MMT

Concentration (ug/L)Length (mm)

0 17 33 70 136 253 0 5 10 15 20 25 30 35 40

XY

XX JT

Vinclozolin F1 Adult Length MED MMT

Concentration (ug/L)Length (mm)

0 17 33 70 136 253 0 5 10 15 20 25 30 35 40

XY

XX JT JTFigure 5, subadult vitellog enin. Gender specific vitellogenin gene expression of subadult medaka in F1 (upper), and F2

(lower) generations. The central line is the mean, the box is the standard deviation and the whisker is the standard error

of the vitellogenin expression. Vitell ogenin gene expression is graphed relative to the gender specific controls.

Significance is indicated by a letter: JT = Jonckheere

- Terpstra. Vinclozolin F1 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 17 33 70 136 253 0 500 1000 1500 2000 2500 3000 3500

XY

XX

Vinclozolin F2 Subadult Vitellogenin MED MMT

Concentration (ug/L)Vtg mRNA Relative to Controls

0 17 33 70 136 0 50 100 150 200 250 300 350 400 450

XY

XX

JTFigure 6, subadult secondary sex characters. Gender specific secondary sex characters (SSC) of sub adult medaka in F1

(upper), and F2 (lower ) generations after exposure to vinclozolin . The central line is the mean, the box is the standard

deviation and the whisker is the standard error of the number of anal fin papillae

, that is the SSC. JT = significantly

different from controls as determined by a Jonckheere

- Terpstra trend test, respectively. Vinclozolin F1 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 17 33 70 136 253 0 20 40 60 80 100 120 140

XY

XX

JT

JT

Vinclozolin F2 Subadult SSC MED MMT

Concentration (ug/L)Anal Fin Papillae (#/fin)

0 17 33 70 136 0 20 40 60 80 100 120 140

XY

XX

JT

JTTable 4. Significant pathologies as indicated by RSCABS in subadult XY medaka of the vinclozolin MED MMT

F1 XY F2 XY

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Testicular Degeneration 70 μg/L 1 70 μg/L 1

- - - -

Interstitial Cell

Hypertrophy - - - - 70 μg/L 1 70 μg/L 1KIDNEY Tubule Mineralization 70 μg/L 1 70 μg/L 1 70 μg/L 2 70 μg/L 2

Glomeruli Protein 33 μg/L 1 33 μg/L 1

- - - -

Tu b Epith Eosinophilia 17 μg/L 3 17 μg/L 3 70 μg/L 3 70 μg/L 3

Tubule Cellular Debris/ Casts

- - - - 70 μg/L 1 70 μg/L 1

Table 5

. Significant pathologies as indicated by RSCABS in subadult XX medaka of the vinclozolin MED MMT

F1 XX F2 XX

LOEC Highest Severity LOEC Highest Severity

Conc Severity Conc Severity Conc Severity Conc Severity

GONAD Increase Lobe Separation 70 μg/L 2 70 μg/L 2 70 μg/L 1 136 μg/L 2

LIVER Basophilia

- - - - 17 μg/L 1 17 μg/L 1Figure 7,subadult weight. Gender specific weights of subadult (9 wpf) medaka in F1 (upper) and F2 (lower) generations

after exposure to vinclozolin. The central line is the mean, the box is the standard deviation and the whisker is the

standard error of the weight measured in mg. Vinclozolin F2 Subadult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 17 33 70 136 0 50 100 150 200 250 300 350

XY

XX Vinclozolin F1 Subadult Weight MED MMT

Concentration (ug/L)Body Weight (mg)

0 17 33 70 136 253 0 50 100 150 200 250 300 350

XY

XXFigure 8, subadult length. Gender specific lengths of subadult (9 wpf) medaka in F1 (upper) and F2 (lower) generations

after exposure to vinclozolin. The central line is the mean, the box is the standard deviation and the whisker is the

standard error of the weight measured in mg. Vinclozolin F1 Subadult Length MED MMT

Concentration (ug/L)Length (mm)

0 17 33 70 136 253 0 5 10 15 20 25 30

XY

XX

Vinclozolin F2 Subadult Length MED MMT

Concentration (ug/L)Length (mm)

0 17 33 70 136 0 5 10 15 20 25 30

XY

XXTable 6. Early life survival (hatch to 3 wpf)

Survival (mean % )

Treatment

Generation control 17 μg/L 33 μg/L 70 μg/L 136 μg/L 253 μg/L

F1 84 82 73 68 56 25

F2 74 83 66 52 68 X

Table 7. Subadult survival (3 wpf to 9 wpf)

Survival (mean % )

Treatment

Generation control 17 μg/L 33 μg/L 70 μg/L 136 μg/L 253 μg/L

F1 99 100 99 100 100 100

F2 100 100 100 100 99 X

Table 8. Hatch

Hatch (mean % )

Treatment

Generation control 17 μg/L 33 μg/L 70 μg/L 136 μg/L 253 μg/L

F1 97 76 89 83 94 65

F2 83 88 91 87 96 X