## 1 Summary Report of

Task Order #32: Medaka Multigeneration Test Interlaboratory Test Evaluation

Prepared by

Battelle

- Pacific Northwest Division

Marine Research Operations

1529 West Sequim Bay Road

Sequim, Washington 98382

May 2013

## 2 SIGNATURE PAGE

Submitted by: Battelle

- Pacific Northwest Division

Marine Research Operations

1529 West Sequim Bay Road

Sequim, Washington 98382

Prepared by:

____________________________________________________________ Irvin R. Schultz, Ph.D. Date

Task Order Project Manager

Battelle

- Pacific Northwest Division

____________________________________________________________ Valerie Cullinan, Ph.D. Date

Statistician

Battelle

- Pacific Northwest Division

Study Title: Medaka Multigeneration Test Interlaboratory Test Evaluation

The final report has been re viewed by Quality Assurance.

__________________________________ ____________________ Mary E. Lynn, M.S., RQAP

- GLP Date

Quality Assurance

## 3 Table of Contents

1.0 Introduction

2.0 Quality Assurance

3.0 Modification of the Test Guidelines

4.0 Chemical Exposure Characterization

5.0 Endpoint Evaluation

6.0 Results

6.1 Control Data Correlation and E ndpoint Variability

6.2 Summary of Individual Studies

7 .0 Discussion

8 .0 References

## 4 1.0 Introduction

The U.S. Environmental Protection Agency (U.S. EPA) is implementing the Endocrine Disruptor

Screening Program (EDSP). To support this program, U.S. EPA contracted Battelle to assist in

developing, standardizing, and validating ecotoxicological test s for i dentifying and

characterizing endocrine effects through exposure to pesticides, industrial chemicals, and

environmental contaminants. The EDSP has completed validation of a battery of Tier 1 assays to

identify chemicals that have the potential to interact with the endocrine system, and is pursuing

validation of longer term Tier 2 assays to identify adverse endocrine effects and provide dose

response information needed for risk assessment

. One of the longer term Tier 2 assays being

considered is the Medaka Multi

- Generation Test (MMT). A fish multi

- generation test method

for studying the effects of endocrine disruptive chemicals ( EDCs ) is relevant to assessing the

adverse consequences of chemicals indicated to be endocrine active in Tier 1 assays. As part of

the validation of the MMT test method, the reproducibility of this method must be

demonstrated. Interlaboratory studies evaluate the comparability of test responses across

multiple laboratories and are necessary to fully evaluate the practical transfe rability of the

protocol and quantitative reproducibility of the method.

The MMT method is intended to detect adverse responses in medaka exposed to vertebrate

relevant endocrine

- active compounds. For T ask Order 3 2 (TO32) the selected test chemical was

vinclozolin, which is considered to be an androgen antagonist. Th is chemical ha s tested positive

in the EDSP Tier 1 fish assay and would therefore be a candidate for EDSP Tier 2 testing.

Important attributes of the MMT are monitoring survival, development and growth during

continuous chemical exposure from the F 0



## - to F 2



## - medaka generations

. R eproduction is

measured only in the F 1



## - medaka generation. These non

- specific effects were augmented by

secondary mechanistic biomarker responses (i.e., hepatic vitellogenin [VTG] gene expression

, and histology )

. The genetic sex of the fish was also measured by PCR methods. The

measurements of survival, development, growth, reproduction, and s econdary mechanistic

biomarker responses are indicative of potential adverse effects on population relevant parameters

and potential endocrine disrupter activity for this species and aquatic vertebrates in general

.

The purpose of this report is to summa rize the results of each individual study conducted under

TO 32 ( ABC Labs 201 3

, Maxxam 201 3 and Fort 201 3 ), note any deviations from the expected

nominal vinclozolin dosing levels, compare the various endpoints for sensitivity among the labs

and discuss th e results relative to other findings with medaka and other fishes in the peer

reviewed literature.

## 5 2.0 Quality Assurance

Each participating l aborator y (ABC, Fort and Maxxam) Quality Assurance (QA) personnel

monitored the conduct of the studies as outli ned in the QAPP and their respective standard

operating procedures. The results of the audits (technical systems audits and audits of data

quality) were reported to each labs s tudy d irector and m anagement as required. Responses to any

audit findings were provided by lab study personnel and were reviewed by QA. Completed audit

reports were provided to the Battelle QA for this task order (Windward Company). The audit

reports were reviewed for data and study qua lity issues and were summarized in monthly reports

to the U.S. EPA.

No data quality issues were identified that would have a significant impact to the conduct,

reporting or conclusions of the studies. The individual lab report s detail any amendments and /or

deviations to the study protocol, including assessing the potential effects on the study. Although

no data quality issues were identified, it is important to note that each lab had certain aspects of

the study fail to meet expectations, which may indi rectly impact conclusions made from the

study. These are listed below by lab:

ABC Labs

There was an unusually high percentage of F1 and F2 histologically male and female medaka

that did not match their sexual genotype (see Tables 1 and 2, pages 6 and 9 o f ABC statistics

report). This included control medaka, which had genotype

– histological agreement in 50

- 56%

of F1 and 86

- 81% of F2 fish (male, female percentages) for which both endpoints were available

for comparison. Although genotype was used to so rt fish for statistical analysis, for some fish

genotype was not available and the histological sex used. Thus, it’s possible the genotype of

these latter fish may have been different from their histological sex. Also, during assessment of

F1 reproductio n, several breeding pairs including three in the control treatment, were discovered

to be of the same phenotypic sex despite the presence of a male / female genotypic pair. This

reduced the number of available replicates for fecundity evaluation and the overall statistical

power of fecundity and embryo viability assessments.

Fort Labs

There was an unusually high percentage of F1 males and F2 females whose histological sex did

not match their sexual genotype (see Tables 1 and 2, pages 6 and 10 of Fort st atistics report).

This included 77% agreement for F2 females and 95% agreement for F2 males. Also, the F2

medaka including control fish were exceptionally undersized at study completion. The mean wet

weights varied between 0.032

– 0.054 g for females an d 0.0285

– 0.049 g for males. The small

size indicated a lack of normal growth and development and is the most likely reason for an

inability to observe anal fin papillae on all F2 fish.

Maxxam Labs

Approximately 6% of the F1 medaka had disagreement bet ween sexual genotype and

histological characterization of the gonads. Anal fins removed from the F1 fish deteriorated

during storage to the extent that quantitative assessment of anal fin papillae was judged

unreliable. Thus, there is no F1 anal fin papi llae data for analysis.

## 6 << This summary report will be reviewed by Battelle QA. >>

## 7 3.0 Modification of the Test Guidelines

There were no modifications made to the guidelines other than minor changes documented as

amendments in each labs report.

## 8 4.0 Chemical Exposure Characterization

The chemical concentration was analyzed weekly during each of the three studies (see ABC

201 3

, Fort 2013

, Maxxam 2013 ). The mean concentration during the exposure period along with

t he coefficient of variation (CV) also was determined for each treatment level during the

exposure to evaluate how well each dose level was maintained (Table 1). Figures 1 through 3

illustrate nominal and measured concentrations of vinclozolin during the MMT s tudy for each

lab

. Dose overlap did not appear to occur during the ABC and Maxxam studies. The week

- to

week variability in measured levels was higher during the Fort lab study and overlap with the

next higher nominal treatment level occurred several time s on study days 26, 90, 96, 103 and

131. Also, a large proportional decline in all measured concentrations occurred on study days 83,

117 and 138, which resulted in overlap with next lowest nominal treatment level. T here were no

reported problems or failu res with the chemical delivery system during all stud ies

. The stock

solution of vinclozolin was prepared using a saturator column similar in design to that described by

Kahl et al ( 1999 ) . The saturator columns were prepared by each laboratory using vincl ozolin

provided by the chemical repository. The performance of the saturator columns and stability of

vinclozolin appeared to vary among the labs, which likely accounts for inter

- laboratory differences

between nominal and measured levels. To compensate for vinclozolin instability, the chemical

delivery systems at each laboratory were adjusted to levels approximately 130% of the targeted

nominal values. Consequently, for Maxxam and Fort Labs, the measured vinclozolin levels

frequently exceeded the ta rget nominal levels (Table 1, Figures 2 and 3) . The exception was ABC

Labs, where measured levels were consistently below nominal levels for all treatments, averaging

between 61

- 68% of nominal levels. The w ithin

- study variability of test c oncentrations were

relatively stable throughout th e study, with CVs less than 20%. The exceptions were in the Fort

lab study previously mentioned and the ABC Labs level 5 treatment, which had a CV of 29%.

## 9 5.0 Endpoint Evaluation

A completely randomized design was u sed for the medaka two

- generation reproductive tests . A

single chemical delivery system and water table were used to conduct each experiment. There

were four to six replicate exposure aquaria depending on the measured variable for each

chemical concentration. The control data from each experiment were characterized with

descriptive statistics and correlation. Descriptive statistics included the mean treatment response

and the coefficient of variation for each dose. Variables with a sm all ( e.g. <10%) within

treatment group CV tend to require less replication to detect biologically meaningful (10%)

differences with high power. For a given laboratory , statistical significance for each endpoint

was evaluated with the nonparametric Kruskal

- Wallis test to determine whether the median

response from at least one dose was significantly different from the rest (i.e., an H

- statistic with

5 degrees of freedom [df]). For the present analysis, the Kruskal

- Wallis p

- values were used to

indicate param eter sensitivity. The nonparametric test was used instead of a parametric test

because of the small sample size (n ≤ 6) for each treatment group. Tests of normality and

homogeneity of variance that rely on the chi

- square distribution require sample sizes of at least

30 or the number of treatment groups times five (Daniel 1978, Snedecor and Cochran 1980).

Chemical

- dosing regimens were considered classifications of fixed effects (i.e., control = 0, low

dose, mid doses, and high dose specific to each chemic al). The Jonckheere

- Terpstra (JT) test of

ordered alternatives was used for the Fort result on the proportion of F1 fertile eggs.

6.0 Results

6.1 Control Data Correlation and Endpoint Variability

Descriptive statistics for the control data were calculate d for all lab studies and summarized in

Table 2. The measurements involving the weight and length of fish had CVs always less than

20 % and frequently below 10%

. Other endpoints exhibited greater variability. Daily fecundity

and viable egg numbers had CVs generally greater than 3 0% except for Maxxam Labs, which

was less than 14% (Table 2)

. Comparison of c ontrol data from each of the laboratory studies

provide s a measure of the natural variation for each of the variables and is summarized in Table

3

. This analysis indicated that for most endpoints, the control response was not consistent

between laboratories . The median responses from F1 r eproductive measurements

, F1 , F2 length

and weight measurements were all significantly different (p < 0.0 1) between laboratories (Table

3 ). Closer inspection of the results suggest some of the significant inter

- lab variability can be

attributed to the relatively small size at termination of the Fort lab F1 and F2 fish (Table 3). The

small size of Fort labs F1 females appears to have contributed to the high inter

- lab variability in

fecundity. Normalizing F1 fecundity for the body mass of the female ( F1 total eggs / day / g day

84 female weight) eliminated the significant difference (p=0.463, Table 3) . Two endpo ints that

showed greater consistency

, which were not statistically different between labs was F1 and F2

anal fin processes and hepatic expression of VTG

- 1. However, it should be noted that successful

measurement of anal fin processes only occurred for two labs in both the F1 and F2 generation,

limiting inter

- lab comparisons.

6. 2 Summary of Individual Lab Studies

## 10 ABC The statistically significant finding s w ere a decrease in F1 day119 male anal fin processes

observed in the highest treatment (KW p=0.022 ; LOEC = 120 μg/L) and an increase in F1 day

80/84 male body weight (KW= 0.029; Table 4)

. Although not statistically significant, there was

a trend towards decreased numbers of anal fin processes in the F1 day 84 and F2 male fish, and

increasing body weight in day 84 female body weight (Table 4). There was no significant effects

on reproduction, however egg production in the highest vinclozolin treatment level caused a

nearly 50% decreas e in fecundity (Table 4).

Fort The highest vinclozolin treatment cau sed statistically significant increases in female body

weight and length that were observed in F1 day 84 females (LOEC= 120 μg/L; KW=0.004

– 0.006 Table 4). Treatment also caused statistically significant increases in male body weights

measured on F1 day 84 and F2 day 168 samplings (LOEC= 120 μg/L; KW=0.015

– 0.041 Table

4)

. Interestingly, the day 119 body weight and lengths were not affected by treatment in either

sex. The daily cumulative number of eggs produced appeared to decline in a dose

- dependent

m anner but was not significantly different than controls (KW=0.067; Table 4). However, the

proportion of fertile eggs was significantly reduced at the 48 μg/L and higher treatments

(J T =<0.001; Table 4). Although not significantly changed, the F1 female VT G

- 1 expression was

increased in the highest treatment level (KW=0.062; Table 4).

Maxxam These results are remarkable for the near lack of significant effects or trends from

vinclozolin treatment. The most important finding was a significant decrease in F2 male anal fin

papillae observed at both the 120 and 300 μg/L treatments (KW=0.002. Tabl e 4). Other

noteworthy results were a nearly significant increase in F1 female day 119 body weight and

length (KW= 0.054

– 0.66; Table 4).

7 .0 Discussion

This study performed a inter

- laboratory comparison of results using the MMT protocol

and vinclozolin as the test chemical. Vinclozolin is a previously established endocrine disruptor

in vertebrates, with a mode of action (MOA) that is androgen antagonis m. None of the labs

observed significant impacts on overall fecundity nor was a single endpoint signif icantly altered

in a consistent manner across all labs. However, significant changes observed in some

endpoints by each lab appear to be consistent with the expected MOA. For example, both ABC

and Maxxam observed significant decreases in male anal fin p apillae in at least one

- generation.

Also, F1 day 84 or day 119 body weights were generally increased at higher treatment levels

among the labs. In a previous MMT medaka study performed by ABC labs (Task Order 27),

vinclozolin treatment levels that roughl y correspond to levels 8

- 120 μg/L of the present study

primarily caused increases in male GSI and the prevalence of interstitial cells in the testes (Hicks

2012)

. There w ere no significant changes o bserved in fecundity or anal fin papillae in Task

Order 27 . These findings with medaka and the MMT protocol are not inconsistent with the

findings from other studies using different fish species and exposure protocols. It is becoming

increasingly apparent that r esponses of apical endpoints in f ishes exposed to anti

- androgens can

be problematic to characterize due to subtle or inconsistent changes across species. For example,

Makynen et al. (2000) reported that 21 day exposure of fathead minnows to nominal vinclozolin

## 11 levels of 200 and 700 μg/L had no effect on male GSI. In a follow

- on study, Martinovic et al.

(2008) reported that fathead minnows exposed for 21 days to measured vinclozolin

concentrations of 255 and 450 μg/L had decreased fecundity and the male GSI was significantly

increased. In medaka, Kiparissis et al. ( 2003 ) reported that a 100 day vinclozolin (nominally

2500 μg/L) caused a significant reduction in mature spermatozoa in the testes. In guppies, oral

exposure to vinclozolin caused significant changes in male secondary sex cha racteristics (Bayley

et al 2002). A recent study using sexually mature male goldfish reported that nominal

vinclozolin levels of 100, 400 and 800 μg/L significantly altered circulating 11

- ketotestosterone

levels and decreased sperm quality parameters such as milt volume and sperm motility (Hatef et

al. 2012). Thus, some of the vinclozolin findings in TO32 are consistent with an anti

- androgenic

MOA, but more interlaboratory consistency in changes associated with reproduction and male

secondary sex characte ristics at the highest vinclozolin treatment level would have been

expected.

An important attribute of multi

- generational tests such as the MMT is the capability to

detect transgenerational effects or transgenerational sensitization to a chemical. Thus, it can be

valuable to compare responses of the F1 generation with the F 2 generation. Based on the

summary presented in Table 4, there were fewer significant changes in the F2 fish compared to

the F1 fish.

A final aspect of the study that is important t o mention is the surprising frequency of

mismatch between genotype with microscopic / histologic sex of the fish. Although it’s possible

that vinclozolin treatment caused phenotypic reversal in some fish, it is unlikely as a similar

phenomenon was observ ed in control fish from all labs (see Table 5 for a summary of ABC, Fort

labs F1 and F2 controls and Maxxam F1 controls). Among unexposed fish, o nly the Fort Labs

F1 and Maxxam F2 controls had 100% agreement between genotype and histological sex.

However , it is important to note that only a relatively few number of fish were examined

histologically so it does not preclude that more mismatches would have been identified if all fish

were examined microscopically. The frequency of disagreement ranged from 5 0% in ABC F1

males to 95% in Fort F2 males. Among each lab, several instances of both genotypic males and

females were later observed to be of the opposite histological sex

, making it difficult to

generaliz e as to whether a specific type of phenotypic re versal was occurring

. However,

inspection of Table 5 and the individual statistic reports suggest s the F1 fish tended to have a

greater frequency of mismatch than F2 fish. It should also be mentioned that in the previous

MMT medaka study with vinclozolin (Task Order 27, Hicks 2012), 35 out of 272 fish examined

had a histological sex that differed from the phenotypic sex. The majority of these differences

were phenotypic females later identified to be microscopically male and the differences did not

appear to be treatment related, as they occurred throughout all treatment groups (Hicks 2012).

The lack of consistent correlation of sexual genotype with gonad type raises important

questions on how best to interpret and implement genotyping data. In the pres ent study, we

relied on genotype as the overriding determinant of sex. However, at least among control fish, it

can be argued that gonad type is more important, as the gonads are the primary site of sex steroid

production in mature fish. This forms the b asis of relying on secondary sex characteristics as an

endpoint because of the involvement of estrogens and androgens in directing formation of certain

characteristics. For example, the implied assumption is that changes in anal fin papillae reflect

chang es in circulating androgens or their activity (in situations of receptor antagonism) in males

or unnatural stimulation in females. However, if a fish with a female genotype matures with

testes, the presence of anal fin papillae would be expected based on gonad type but unexpected

## 12 based on genotype. If treated as female, this result then skews the values upwards for genotypic

and histologic females (which typically have no papillae) , increasing variability and reducing the

power to detect significant differences among treated fish

. The converse is also true with

respect to males

- including genotypic males that are histological females lower s mean papillae

counts and increase s variability. A good example of this effect can seen in the ABC labs

stati stical analysis of day 84 papillae counts (page 8 of Appendix A, Hicks 2013). Note the high

variability in control values that contribute to a lack of statistical significance. A similar type of

argument can be made for other sex dependent endpoints suc h as VTG.

Our recommendation is to treat sexual genotype as an “endpoint” and rely on

microscopic examination of the gonads as the primary determinant of sex in mature,

reproductively active fish. This would permit calculating the frequency of mismatch in all fish in

all treatments and could be analyzed statistically, hence its use as an endpoint. In situations

where gonad type cannot be unambiguously determined (e.g. ovo

- testes or gross

underdevelopment), special data handling may be required

. Based on experience gained from

TO27 and TO32, spontaneous intersex appears to be extremely rare in medaka, so its occurrence

would more likely be the result of chemical exposures. The full value of genotyping becomes

clear er when combined with microscopic examin ation as any treatment induced changes in sex

reversal will likely become more apparent. It also allows permits greater potential to detect

transgenerational effects on sex reversal. The added burden of requiring labs to assess

microscopic sex determina tion is minimal, when it is performed using a dissecting scope at time

of liver or gonad collection ( if GSI is added to protocol )

.

8 .0 References

Bayley, M., Junge, M., and Baatrup, E. (2002). Exposure of juvenile guppies to three

antiandrogens cause s demasculinization and a reduced sperm count in adult males. Aquatic

Toxicology 56 (4), 227

- 239.

Daniel, Wayne W. 1978. Applied Nonparametric Statistics. Houghton Mifflin Company, Boston,

Massachusetts, 510 pages.

Hatef, A

., Alavi, S

., Mohammad

, H

., Milla, S

., Krist'an, J

., Golshan, M

., Fontaine, P

., Linhart, O

. (2012). Anti

- androgen vinclozolin impairs sperm quality and steroidogenesis in goldfish

. Aquatic Toxicology 122 : 181

- 187

.

Hicks, S.L. 20 12 . : Vinclozolin : The Medaka ( Oryzias latipes ) Full Life

- Cycle Test ABC

Laboratories Study No. 647 60

. Final Report Prepared for U.S. EPA

.

Keating

, J . 20 1 3

. Vinclozolin : The Medaka ( Oryzias latipes ) Full Life

- Cycle Test Maxxam

Laboratories Study No. XXX

. Final Report Prepared for U.S. EPA

.

Fort, D. 20 1 3

. Vinclozolin : The Medaka (Oryzias latipes) Full Life

- Cycle Test. Fort Laboratories

Study No. XXX

. Final Report Prepared for U.S. EPA

.

## 13 Hicks, S.L. 20 1 3 . : Vinclozolin : The Medaka ( Oryzias latipes ) Full Life

- Cycle Test ABC

Laboratories Study No. XXX

. Final Report Prepared for U.S. EPA

.

Kahl, M. D., Russom, C. L., Defoe, D. L., and Hammermeister, D. E. (1999). Saturation units for

use in aquatic bioassays. Chemosphere 39 (3), 539

- 551.

Kiparissis, Y., Metcalfe, T. L., Balch, G. C., and Metcalf, C. D. (2003). Effects of the

antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese

medaka (Oryzias latipes). Aquatic Toxicology 63 (4), 391

- 403.

Makynen, E. A., Kahl, M. D., Jensen, K. M., Tietge, J. E., Wells, K. L., Van der Kraak, G., and

Ankley, G. T. (2000). Effects of the mammalian antiandrogen vinclozolin on development and

reproduction of the fathead minnow (Pimephales promelas). Aquatic Toxicology 48 (4), 4 61

- 475.

Martinovic, D., Blake, L. S., Durhan, E. J., Greene, K. J., Kahl, M. D., Jensen, K. M., Makynen,

E. A., Villeneuve, D. L., and Ankley, G. T. (2008). Reproductive toxicity of vinclozolin in the

fathead minnow: Confirming an anti

- androgenic mode of a ction. Environmental Toxicology and

Chemistry 27 (2), 478

- 488.

Snedecor, G.W. and W.G. Cochran. 1980. Statistical Methods, 6th Edition. The Iowa State

University Press. Ames, Iowa. 593 pages.

## 14 Appendix Tables



## Table 1. Nominal Dose Concentrations, Mean Exposure Dose, Coefficient of Variation, and

Percentage of Nominal Dose for Each Dose Level Tested During Task Order 32

Nominal Units Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

ABC Labs μg a.i. /L 0 8.0 19 48 120 300

Fort Labs μg a.i. /L 0 8.0 19 48 120 300

Maxxam Labs μg a.i. /L 0 8.0 19 48 120 300

Mean Exposure Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

ABC Labs μg a.i. /L <MQL a 

## 4.85 12.6 30.9 81.6 197

Fort Labs μg a.i. /L <MQL b 

## 11.6 25.2 62.0 155 360

Maxxam Labs μg a.i. /L <MQL c 

## 8.9 21.7 52.3 133 333

% CV Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

ABC Labs NA 0.88 1.70 6.21 10.1 29.2

Fort Labs NA 49.9 36.5 30.8 32.8 24.7

Maxxam Labs NA 14.6 10.1 10.9 10.4 8.5

% of Nominal Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

ABC Labs NA 61 66 64 68 66

Fort Labs NA 145 133 129 129 120

Maxxam Labs NA 111 114 109 111 11 1

a 

### Minimum Quantifiable Limit (MQL) = 2.28 μg a.i./L

b 

### Minimum Quantifiable Limit (MQL) = 1.14 μg a.i./L

c 

### Minimum Quantifiable Limit (MQL) = 5.0 μg a.i./L

## 15 Ta bl e 2. Control Data Descriptive Statistics For the F1 and F2 Generations

Variable Lab N Mean Standard

Deviation 

## Minim

um Medi

an Maxim

um % CV

F1 Total Eggs/Day ABC 3 27.1 8.24 19 26.81 35.48 30%

Fort 6 10.02 3.32 6.24 8.71 15.19 33%

Maxxam 6 34.71 4.97 26.24 35.36 39.6 14%

F1 Viable Eggs/Day ABC 3 21.73 12.02 9.14 22.95 33.1 55%

Fort 6 9.68 3.34 5.62 8.5 14.81 35%

Maxxam 6 33.67 4.65 25.9 33.79 39.2 14%

F1 Proportion Viable ABC 3 0.757 0.242 0.481 0.856 0.933 32%

Fort 6 0.9614 0.0298 0.9008 0.973 0.978 3%

Maxxam 6 0.970 9 0.01867 0.94 70 0.974 0.9899 2%

F1

- Female Day

80/84 Weight (g) ABC 6 0.160 8 0.02392 0.134 8 0.151 0.1965 15%

Fort 4 0.088 4 0.01458 0.074 3 0.085 0.1087

5 17%

Maxxam 6 0.2072 0.0253 0.1863 0.19 8 0.2553 12%

F1 Female Day

80/84 Length (mm) ABC 0

-- -- -- -- -- --

Fort 4 19.799 1.061 18.75 19.8 5 20.75 5%

Maxxam 6 26.625 0.997 25 26.75 28 4%

F1

- Female Day

80/84 copies

VTG/ng RNA ABC 5 22289 14469 9012 1444 42082 65%

Fort 2 1195 1679 7 1195 2382 141%

Maxxam 4 17470 16180 4602 1206 41157 93%

F1 Male Day 80/84

Weight (g) ABC 6 0.127 2 0.01516 0.1135 0.123 0.1568

3 12%

Fort 6 0.1066 0.00807 0.0912 0.109 0.1135

3 8%

Maxxam 6 0.1986 0.01154 0.1864 0.198 0.2109

6 6%

F1 Male Day 80/84

Length (mm) ABC 0

-- -- -- -- -- --

Fort 6 21.776 0.95 20.06 22.22 22.475 4%

Maxxam 6 26.006 0.832 24.333 26.25 26.6 3%

F1 Male Day 84

Total Processes ABC 6 26.02 21.09 12.25 15.98 67.5 81%

Fort 6 14.73 12.15 0 18.42 28.43 82%

Maxxam 0

-- -- -- -- -- --

## 16 Variable Lab N Mean Standard

Deviation 

## Minim

um Medi

an Maxim

um % CV

F1 Male Day 80/84

copies VTG/ng RNA ABC 5 2.25 2.44 0.33 1.21 6.32 108%

Fort 2 27 22.6 11 27 43 84%

Maxxam 6 1850 4308 1 9 10635 233%

F1 Female Day 119

Weight (g) ABC 0

-- -- -- -- -- --

Fort 6 0.1916 0.0417 0.1417 0.18 9 0.2499 22%

Maxxam 5 0.4187 0.0644 0.3481 0.460 0.4705 15%

F1 Female Day 119

Length (mm) ABC 0

-- -- -- -- -- --

Fort 6 27.083 1.594 25.6 26.7 29.5 6%

Maxxam 5 32.4 1.342 31 33 34 4%

F1 Male Day 119

Weight (g) ABC 0

-- -- -- -- -- --

Fort 6 0.227 0.0268 0.1988 0.226 0.2668 12%

Maxxam 6 0.3887 0.0598 0.298 0.40 9 0.447 15%

F1 Male Day 119

Length (mm) ABC 0

-- -- -- -- -- --

Fort 6 29.383 1.809 26.2 30.2 31 6%

Maxxam 6 32.333 1.033 31 33 33 3%

F1 Male Day 119

Total Processes ABC 6 73.5 13.8 50 74.75 89.5 19%

Fort 6 53.33 6.47 47 53 64 12%

Maxxam 0

-- -- -- -- -- --

F2 Control Data Descriptive Statistics

Variable Lab N Mean Standar

d

Deviati

on Minim

um Medi

an Maxim

um CV

F2 Female

Termination Weight

(g) ABC 6 0.211 0.023 0.173 0.215 0.242 11

%

Fort 6 0.034 0.005 0.027 0.033 0.040 14

%

Maxxam 6 0.226 0.012 0.214 0.225 0.246 5%

F2 Female

Termination Length

(mm) ABC 0

-- -- -- -- -- --

Fort 6 15.4 1.35 12.9 16.1 16.3 9%

Maxxam 6 27.3 0.464 26.8 27.4 27.8 2%

F2 Female ABC 6 10.1 17.9 0.00 0.00 44.0 177

## 17 Variable Lab N Mean Standar

d

Deviati

on Minim

um Medi

an Maxim

um CV

Termination Total

Processes %

Fort 0

-- -- -- -- -- --

Maxxam 6 1.43 3.50 0.00 0.00 8.57 245

%

F2 Female

Termination copies

VTG/ng RNA ABC 2 23650 1768 22400 2365 24900 7%

Fort 2 0.50 0.00 0.50 0.50 0.50 0%

Maxxam 4 43273 22471 17148 420 1 71925 52

%

F2 Male

Termination Weight

(g) ABC 6 0.193 0.024 0.171 0.186 0.231 13

%

Fort 6 0.028 0.010 0.015 0.032 0.040 36

%

Maxxam 6 0.222 0.010 0.201 0.225 0.228 5%

F2 Male

Termination Length

(mm) ABC 0

-- -- -- -- -- --

Fort 6 14.7 1.99 12.0 15.3 16.6 14

%

Maxxam 6 28.1 0.54 27.3 28.0 28.8 2%

F2 Male

Termination Total

Processes ABC 6 52.9 35.7 0.0 58.9 91.4 67

%

Fort 0

-- -- -- -- -- --

Maxxam 6 59.1 4.9 54.8 57.6 67.4 8%

F2 Male

Termination copies

VTG/ng RNA ABC 3 1.22 1.58 0.00 0.67 3.00 129

%

Fort 2 2.50 3.54 0.00 2.50 5.00 142

%

Maxxam 4 1.75 1.26 0.00 2.00 3.00 72

%

## 18 Table 3

. Median Control Response by Chemical and the Kruskal

- Wallis Test of Equal Medians



## Variable ABC Fort Maxxam Kruskal

- Wallis p

- value



## F1 Total Eggs/Day 27.1 10.02 34.71 0.005



## F1 Total Eggs/Day / day 84 female weight 170.4 121.5 169.4 0.463



## F1 Viable Eggs/Day 21.73 9.68 33.67 0.005



## F1 Proportion Viable 0.757 0.9614 0.97086 0.044



## F1

- Female Day 80/84 Weight (g) 0.16 1 0.08 9 0.207 0.002



## F1 Female Day 80/84 Length (mm)

-- 19.799 26.625 0.01



## F1 Fem ale Day 84 Total Processes 7.8 0

--

F1

- Female Day 80/84 copies VTG/ng RNA 22289 1195 17470 0.076



## F1 Male Day 80/84 Weight (g) 0.127 0.10 7 0.19 9 0.001



## F1 Male Day 80/84 Length (mm)

-- 21.7 8 26.0 1 0.004



## F1 Male Day 84 Total Processes 26.02 14.73

-- 0.423



## F1 Male Day 80/84 copies VTG/ng RNA 2.25 27 1850 0.075



## F1 Female Day 119 Weight (g)

-- 0.19 2 0.41 9 0.006



## F1 Female Day 119 Length (mm)

-- 27.08 32.4 0.006



## F1 Fe ale Day 119 Total Processes 0 0

-- F1 Male Day 119 Weight (g)

-- 0.227 0.38 9 0.004



## F1 Male Day 119 Length (mm)

-- 29.38 32.33 0.005



## F1 Male Day 119 Total Processes 73.5 53.33

-- 0.025



## F2 Female Termination Weight (g) 0.211 0.034 0.226 0.002



## F2 Female Termination Length (mm)

-- 15.4 27.3 0.004



## F2 Female Termination Total Processes 10.1

-- 1.43 0.40



## F2 Female Termination copies VTG/ng RNA 23650 0.5 43273 0.103



## F2 Male Termination Weight (g) 0.193 0.028 0.222 0.002



## F2 Male Termination Length (mm)

-- 14.7 28.1 0.004



## F2 Male Termination Total Processes 52.9

-- 59.1 1.0



## F2 Male Termination copies VTG/ng RNA 1.22 2.50 1.75 0.912

## 19 Table 4. Descriptive Statistics for F1 and F2 Generation Exposure to Vinclozolin.

F1 Table

Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

F1

- Female

Day 80/84

Wet Weight (g) 0 6 0.16 15% 4 0.09 17% 6 0.21 12%

8 5 0.16 13% 6 0.12 18% 6 0.2 0 6%

19 5 0.15 32% 4 0.11 33% 6 0.2 0 6%

48 6 0.16 24% 6 0.12 9% 6 0.21 6%

120 6 0.2 9% 6 0.13 35% 6 0.19 19%

300 6 0.18 7% 6 0.18 14% 6 0.21 10%

Kruskal

- Wallis p

- value 0.091 0.006 0.719

F1

- Female

Day 80/84

Length (mm) 0

-- -- -- 4 19.8 5% 6 26.6 4%

8

-- -- -- 6 21.7 8% 6 26.4 3%

19

-- -- -- 4 21.2 12% 6 25.7 4%

48

-- -- -- 6 22.2 5% 6 25.8 4%

120

-- -- -- 6 20.1 14% 6 25.5 4%

300

-- -- -- 6 24.4 5% 6 26.1 2%

Kruskal

- Wallis p

- value

-- 0.004 0.281

F1

- Male Day 80/84

Wet Weight (g) 0 6 0.13 12% 6 0.11 8% 6 0.2 6%

8 6 0.14 18% 6 0.13 10% 6 0.2 7%

19 6 0.16 20% 6 0.12 10% 6 0.19 11%

48 4 0.13 13% 6 0.12 10% 6 0.18 11%

120 6 0.14 9% 6 0.13 16% 6 0.2 7%

300 6 0.15 15% 6 0.14 18% 6 0.2 11%

Kruskal

- Wallis p

- value 0.0294 0.015 0.398

F1

- Male

Day 80/84

Length (mm) 0

-- -- -- 6 21.8 4% 6 26 3%

8

-- -- -- 6 23 4% 6 26.1 3%

19

-- -- -- 6 22.3 5% 6 26 2%

48

-- -- -- 6 23 4% 6 26.2 3%

120

-- -- -- 6 22.3 7% 6 26.5 2%

300

-- -- -- 6 23.3 7% 6 26.2 5%

Kruskal

- Wallis p

- value

-- 0.286 0.761

F1

- Cumulative

Number of

Eggs/Day 0 3 27.1 30% 6 10.0 33% 6 34.7 14%

8 4 23.7 47% 6 10.4 10% 6 28.4 37%

19 5 23.0 23% 6 7.76 55% 6 34.1 12%

48 6 23.8 19% 6 8.79 17% 6 33.2 28%

120 6 26.0 27% 6 7.24 19% 6 28.2 47%

## 20 Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

300 4 14.4 34% 6 6.66 51% 6 36.6 22%

Kruskal

- Wallis p

- value 0.195 0.067 0.602

F1

- Cumulative

Number of Fertile

Eggs/Day 0 3 21.7 55% 6 9.68 35% 6 33.7 14%

8 4 19.4 59% 6 9.92 9% 6 26.6 45%

19 5 14.7 57% 6 7.36 55% 6 33.1 11%

48 6 19.7 35% 6 8.14 17% 6 30.5 33%

120 6 22.3 41% 6 6.46 21% 6 27.5 52%

300 4 8.68 60% 6 5.40 52% 6 33.6 29%

Kruskal

- Wallis p

- value 0.223 0.021 0.757

F1

- Proportion

Fertile 0 3 0.76 32% 6 0.96 3% 6 0.97 2%

8 4 0.75 35% 6 0.95 2% 6 0.88 25%

19 5 0.61 46% 6 0.93 6% 6 0.97 2%

48 6 0.81 23% 6 0.93 1% 6 0.91 8%

120 6 0.81 28% 6 0.89 3% 6 0.88 30%

300 4 0.55 38% 6 0.81 8% 6 0.9 9%

Kruskal

- Wallis or JT a p

- value 0.240 < 0.001 a 0.173

F1

- Male

Day 84

Total Anal Fin

Papillary Processes 0 6 26 81% 6 14.7 82%

-- -- --

8 6 32.1 50% 6 27.3 53%

-- -- --

19 6 21.1 36% 6 26.7 41%

-- -- --

48 4 32 24% 6 31.6 26%

-- -- --

120 6 26.7 18% 6 23.9 61%

-- -- --

300 6 16.5 33% 6 23.4 22%

-- -- --

Kruskal

- Wallis p

- value 0.078 0.381

--

F1

- Female

Day 80/84

Vtg (ng RNA) 0 5 22289 65% 2 1195 141% 4 17470 93%

8 0

-- -- 0

-- -- 0

-- --

19 0

-- -- 0

-- -- 0

-- --

48 0

-- -- 0

-- -- 0

-- --

120 3 11654 35% 3 2148 71% 2 8662 32%

300 3 19935 23% 3 8923 57% 5 22431 76%

Kruskal

- Wallis p

- value 0.273 0.062 0.404

F1

- Male

Day 80/84

Vtg (ng RNA) 0 5 2.25 108% 2 27 84% 6 1850 233%

8 0

-- -- 0

-- -- 0

-- --

19 0

-- -- 0

-- -- 0

-- --

48 0

-- -- 0

-- -- 0

-- --

120 3 0.249 69% 2 8.5 25% 2 2.25 141%

## 21 Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

300 2 3431 141% 3 25.7 30% 4 16.88 113%

Kruskal

- Wallis p

- value 0.152 0.153 0.319

F1

- Female

Day 119

Wet Weight (g) 0

-- -- -- 6 1.92 22% 5 0.42 15%

8

-- -- -- 6 1.87 14% 4 0.44 16%

19

-- -- -- 6 1.91 39% 6 0.39 6%

48

-- -- -- 6 1.78 18% 6 0.47 12%

120

-- -- -- 6 1.74 13% 6 0.43 12%

300

-- -- -- 6 2.25 11% 6 0.5 14%

Kruskal

- Wallis p

- value

-- 0.219 0.054

F1

- Female

Day 119

Length (mm) 0

-- -- -- 6 27.1 6% 5 32.4 4%

8

-- -- -- 6 26.6 4% 4 33 2%

19

-- -- -- 6 26 14% 6 33.5 3%

48

-- -- -- 6 26.3 5% 6 33.5 5%

120

-- -- -- 6 25.7 5% 6 33.3 4%

300

-- -- -- 6 28.1 7% 6 35 4%

Kruskal

- Wallis p

- value

-- 0.412 0.066

F1

- Male

Day 119

Wet Weight (g) 0

-- -- -- 6 2.27 12% 6 0.39 15%

8

-- -- -- 6 2.48 21% 5 0.36 20%

19

-- -- -- 6 2.74 20% 6 0.33 21%

48

-- -- -- 6 2.34 14% 6 0.37 25%

120

-- -- -- 6 2.54 28% 5 0.41 15%

300

-- -- -- 6 2.6 22% 6 0.37 11%

Kruskal

- Wallis p

- value

-- 0.639 0.340

F1

- Male

Day 119

Length (mm) 0

-- -- -- 6 29.4 6% 6 32.3 3%

8

-- -- -- 6 30 7% 5 33 6%

19

-- -- -- 6 30.3 7% 6 33 5%

48

-- -- -- 6 30 4% 6 32.5 7%

120

-- -- -- 6 30 10% 5 34.6 6%

300

-- -- -- 6 29.8 9% 6 32.8 3%

Kruskal

- Wallis p

- value

-- 0.943 0.388

F1

- Male

Day 119

Total Anal Fin

Papillary Processes 0 6 73.5 19% 6 53.3 12%

-- -- --

8 5 65.7 8% 6 52.7 8%

-- -- --

19 5 73.3 12% 6 54 11%

-- -- --

48 6 58.5 23% 6 50.7 8%

-- -- --

120 6 53.2 37% 6 51.2 10%

-- -- --

300 6 49.8 17% 6 53.3 15%

-- -- --

## 22 Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

Kruskal

- Wallis p

- value 0.022 0.853

--



## F2 Table

Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

F2

- Female Day

168Wet Weight (g) 0 6 0.21 11% 6 0.033

8 14% 6 0.23 5%

8 6 0.23 9% 6 0.032 19% 6 0.22 4%

19 6 0.2 10% 6 0.054

4 30% 6 0.21 10%

48 6 0.21 9% 6 0.034

2 32% 6 0.22 5%

120 6 0.21 15% 6 0.044 30% 6 0.23 4%

300 3 0.22 3% 6 0.034

6 40% 6 0.21 6%

Kruskal

- Wallis p

- value 0.414 0.137 0.497

F2

- FemaleDay

168Length (mm) 0

-- -- -- 6 15.4 9% 6 27.3 2%

8

-- -- -- 6 14.7 7% 6 27.5 3%

19

-- -- -- 6 16.4 13% 6 27 6%

48

-- -- -- 6 15.6 9% 6 27.3 3%

120

-- -- -- 6 16.6 9% 6 27.7 2%

300

-- -- -- 6 15.4 13% 6 27 2%

Kruskal

- Wallis p

- value

-- 0.486 0.617

F2

- Male Day 168Wet

Weight (g) 0 6 0.19 13% 6 0.028

5 36% 6 0.22 5%

8 6 0.2 13% 6 0.031

9 46% 6 0.21 11%

19 6 0.17 8% 6 0.037

4 19% 6 0.19 10%

48 6 0.2 12% 6 0.050

7 31% 6 0.21 6%

120 6 0.2 15% 6 0.049

2 47% 6 0.21 3%

300 4 0.19 11% 6 0.049 24% 6 0.21 9%

Kruskal

- Wallis p

- value 0.183 0.041 0.079

F2

- MaleDay

168Length (mm) 0

-- -- -- 6 14.7 14% 6 28.1 2%

8

-- -- -- 6 14.8 13% 6 27.7 3%

19

-- -- -- 6 15.2 6% 6 26.8 4%

48

-- -- -- 6 17.4 11% 6 27.5 2%

## 23 Variable Treatment ABC Fort Maxxam

N Mean CV N Mean CV N Mean CV

120

-- -- -- 6 17.2 18% 6 27.1 2%

300

-- -- -- 6 16.6 4% 6 27.5 3%

Kruskal

- Wallis p

- value

-- 0.090 0.096

F2

- FemaleTotal Anal

FinPapillaryProcesses 0 6 10.1 177%

-- -- -- 6 1.43 245%

8 6 16.6 111%

-- -- -- 6 0 0%

19 6 6.8 126%

-- -- -- 6 0 0%

48 6 2.04 157%

-- -- -- 6 0 0%

120 6 8.31 143%

-- -- -- 6 2.25 155%

300 3 0 0%

-- -- -- 6 0 0%

Kruskal

- Wallis p

- value 0.335

-- --

F2

- MaleTotal Anal

FinPapillaryProcesses 0 6 52.9 67%

-- -- -- 6 59.1 8%

8 6 40.2 62%

-- -- -- 6 39.7 33%

19 6 60.7 12%

-- -- -- 6 40 41%

48 6 35.9 38%

-- -- -- 6 37.8 21%

120 6 31 69%

-- -- -- 6 29.5 59%

300 4 29.5 25%

-- -- -- 6 21.3 36%

Kruskal

- Wallis p

- value 0.116

-- 0.002

F2

- Female Vtg(ng

RNA) 0 2 23650 7% 2 0.5 0% 4 43273 52%

8 0

-- -- 0

-- -- 0

-- --

19 0

-- -- 0

-- -- 0

-- --

48 0

-- -- 0

-- -- 0

-- --

120 2 34329 61% 2 397 132% 5 26284 109%

300 2 42896 102% 2 12.5 142% 6 52995 144%

Kruskal

- Wallis p

- value 1 .00 0.246 0.418

F2

- Male Vtg(ng RNA) 0 3 1.22 129% 2 2.5 142% 4 1.75 72%

8 0

-- -- 0

-- -- 0

-- --

19 0

-- -- 0

-- -- 0

-- --

48 0

-- -- 0

-- -- 0

-- --

120 4 0.46 137% 3 0.667 87% 6 1 127%

300 4 0.25 200% 4 0.25 200% 5 6 122%

Kruskal

- Wallis p

- value 0.528 0.543 0.277



## Table 5 Genotype and histological sex among control fish

## 24 ABC Control Data

Genotype

F1 Day 84 Histology Total

F M Not

Determined

Female (F) 9 7 1 17

Male (M) 8 8 2 18

Not Determined 7 6 0 13



## Female agreement = 56% Male agreement = 50%

Genotype

F2 Study Day 168 Histology

Total F M Not

Determined

Female (F) 13 3 2 18

Male (M) 4 24 0 28

Not Determined 0 0 0 0



## Female agreement = 81% Male agreement = 86%

Fort Control Data

Genotype

F1 Day 84 Histology

Total F M Not

Determined

Female (F) 12 0 1 13

Male (M) 0 12 21 33

Not Determined 0 0 1 1



## Female agreement = 100%

Male agreement = 100%

Genotype

F1 Day 119 Histology

Total F M Not

Determined

Female (F) 5 0 0 5

Male (M) 0 6 0 6

## 25 Questionable F 1 0 0 1



## Female agreement = 100% Male agreement = 100%

Genotype

F2 Study Day 168 Histology

Total F M Not

Determined

Female (F) 48 14 101 163

Male (M) 3 56 91 150

Not Determined

or Missing 2 1 7 10



## Female agreement = 77% Male agreement = 95%

Maxxam Control Data

Genotype

F1 Day 80 Histology

Total F M Not

Determined

Female (F) 9 2 6 17

Male (M) 2 11 14 27

Not Determined 0 0 2 2



## Female agreement = 82%

Male agreement = 85%

## 26

## 27

## 28