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 reviewed 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 Endpoint 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 tests for identifying 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 transferability of the 

protocol and quantitative reproducibility of the method. 

The MMT method is intended to detect adverse responses in medaka exposed to vertebraterelevant endocrine-active compounds. For Task Order 32 (TO32) the selected test chemical was 

vinclozolin, which is considered to be an androgen antagonist. This chemical has 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 F0- to F2-medaka generations. Reproduction is

measured only in the F1-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 secondary 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 summarize the results of each individual study conducted under 

TO 32 (ABC Labs 2013, Maxxam 2013 and Fort 2013), note any deviations from the expected 

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

and discuss the results relative to other findings with medaka and other fishes in the peerreviewed literature.
5

2.0 Quality Assurance

Each participating laboratory (ABC, Fort and Maxxam) Quality Assurance (QA) personnel 

monitored the conduct of the studies as outlined 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 study director and management 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 quality 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 reports 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 indirectly 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 of 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 sort 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 reproduction, 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 statistics 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 and 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 between 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 papillae 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 

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

the 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 study for each 

lab. Dose overlap did not appear to occur during the ABC and Maxxam studies. The week-toweek variability in measured levels was higher during the Fort lab study and overlap with the 

next higher nominal treatment level occurred several times 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. There were no 

reported problems or failures with the chemical delivery system during all studies. 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 vinclozolin 

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 target 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 within-study variability of test concentrations were 

relatively stable throughout the 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 used 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 small (e.g. <10%) withintreatment 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 parameter 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 chemical). 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 calculated 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 30% except for Maxxam Labs, which 

was less than 14% (Table 2). Comparison of control data from each of the laboratory studies

provides 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 reproductive measurements, F1, F2 length 

and weight measurements were all significantly different (p < 0.01) 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 endpoints 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 findings were 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% decrease in fecundity (Table 4).

Fort The highest vinclozolin treatment caused 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 

manner 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 

(JT=<0.001; Table 4). Although not significantly changed, the F1 female VTG-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. Table 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 antagonism. None of the labs 

observed significant impacts on overall fecundity nor was a single endpoint significantly 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 papillae 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 roughly 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 were no significant changes observed 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 responses of apical endpoints in fishes 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 characteristics (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 characteristics 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 F2 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 to 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 observed 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, only 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 50% 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 

generalize as to whether a specific type of phenotypic reversal was occurring. However, 

inspection of Table 5 and the individual statistic reports suggests 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 present 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 basis 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 

changes 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 lowers mean papillae

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

statistical 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 such 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

clearer when combined with microscopic examination 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 determination 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 causes 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. 2012. : Vinclozolin: The Medaka (Oryzias latipes) Full Life-Cycle Test ABC 

Laboratories Study No. 64760. Final Report Prepared for U.S. EPA.

Keating, J. 2013. Vinclozolin: The Medaka (Oryzias latipes) Full Life-Cycle Test Maxxam

Laboratories Study No. XXX. Final Report Prepared for U.S. EPA.

Fort, D. 2013. 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. 2013. : 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), 461-475.

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 action. 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 μga.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 <MQLa 4.85 12.6 30.9 81.6 197

Fort Labs μg a.i. /L <MQLb 11.6 25.2 62.0 155 360

Maxxam Labs μg a.i. /L <MQLc 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 111 114 109 111 111 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

Table 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 0.9709 0.01867 0.9470 0.974 0.9899 2%

F1-Female Day 

80/84 Weight (g)

ABC 6 0.1608 0.02392 0.1348 0.151 0.1965 15%

Fort 4 0.0884 0.01458 0.0743 0.085 0.1087

5 17%

Maxxam 6 0.2072 0.0253 0.1863 0.198 0.2553 12%

F1 Female Day 

80/84 Length (mm)

ABC 0 -- -- -- -- -- --

Fort 4 19.799 1.061 18.75 19.85 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 0.1272 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 0.189 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 0.409 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 4201 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) 0.161 0.089 0.207 0.002

F1 Female Day 80/84 Length (mm) -- 19.799 26.625 0.01

F1 Female 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 0.107 0.199 0.001

F1 Male Day 80/84 Length (mm) -- 21.78 26.01 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) -- 0.192 0.419 0.006

F1 Female Day 119 Length (mm) -- 27.08 32.4 0.006

F1 Feale Day 119 Total Processes 0 0 --

F1 Male Day 119 Weight (g) -- 0.227 0.389 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 0.20 6%

19 5 0.15 32% 4 0.11 33% 6 0.20 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 JTa p-value 0.240 < 0.001a 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

F M Total

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

F M Total

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

F M Total

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

F M Total

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

F M Total

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