Page 1 of 60

 Chemical Safety

 and Pollution Prevention

 (7101)

EPA No. tbd

Draft of December 17, 2014

Endocrine Disruptor 

Screening Program Test 

Guidelines

OCSPP 890.2200:

Medaka Extended One 

Generation Reproduction 

Test (MEOGRT)

PUBLIC REVIEW DRAFT
Public Review Draft of December 17, 2014

Page 2 of 60

NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental 

Protection Agency’s Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing 

pesticides and chemical substances to develop data for submission to the Agency under the Toxic 

Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and 

Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of 

Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines 

issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS 

as part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use the new 

acronym (OCSPP) in their title.

The OCSPP test guidelines serve as a compendium of accepted scientific methodologies and 

protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA and/or 

FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public 

and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the 

FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside 

parties, and the EPA may depart from the guidelines where circumstances warrant and without prior 

notice. At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than mandatory. The 

procedures contained in this guideline are strongly recommended for generating the data that are the 

subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. 

You may propose alternatives to the recommendations described in these guidelines, and the Agency 

will assess them for appropriateness on a case-by-case basis. 

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & Guidelines” on the 

left side navigation menu. You may also access the guidelines in http://www.regulations.gov grouped 

by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and 

EPA-HQ-OPPT-2009-0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the purpose of 

external review. It has not been formally disseminated by the EPA and should not be construed to 

represent any Agency determination or policy. The information correction process under the Agency’s 

Information Quality Guidelines does not apply until this document is formally disseminated by the EPA 

in its final form. This draft document should only be cited or quoted in the context of providing 

comments. 
Public Review Draft of December 17, 2014

Page 3 of 60

OCSPP 890.2200: Medaka Extended One Generation Reproduction Test

(a) Scope.

(1) Applicability. This guideline is intended to meet testing requirements of FIFRA, 

FFDCA, and TSCA.

(2) Background. The Endocrine Disruptor Screening Program (EDSP) reflects a 

two-tiered approach to implement the statutory testing requirements of FFDCA section 

408(p) (21 U.S.C. 346a). In general, EPA intends to use the data collected under the 

EDSP, along with other information, to evaluate potential risk to human health or the 

environment that may occur from a putative endocrine active substance. 

This Test Guideline is intended to be used in Tier 2 of the EDSP screening program as a 

definitive test of the effects of a test substance as a potential endocrine disrupting 

chemical (EDC) on growth, survival, and reproduction. Additional endpoints are also 

included in the guidelines that may provide information regarding the general Adverse 

Outcome Pathway(s) (AOPs) affected by the test substance. 

The test substance will have undergone testing through the battery of Tier 1 screening 

assays (OCSPP 890 series) and the weight of evidence analysis of other scientifically 

relevant information as prescribed in the EDSP and have been found to potentially 

interact with the estrogen (E), androgen (A), or thyroid (T) hormone systems. At which 

time, the Agency will have determined the need to proceed with Tier 2 testing including 

this guideline. Tier 2 testing is designed to identify any adverse apical effects caused by 

the test substance, and to establish a quantitative dose-response relationship for these 

effects.

(b) Purpose. 

The purpose of definitive Tier 2 testing is to determine whether a test substance adversely 

affects a test organism through endocrine-mediated pathways, and to quantitatively evaluate 

these effects. In addition, the tests must include exposure during the most sensitive life stages, 

and provide the opportunity for identification of dose-response effects.

The Medaka Extended One Generation Reproduction Test (MEOGRT) characterizes the nature, 

likelihood, and dose-response relationship of apical adverse outcomes from potential endocrine 

disruption via E, A, and/or T pathways. In combination with other selected ecotoxicity EDSP 

Tier 2 tests, the previously completed EDSP Tier 1 screening battery, and other relevant 

information (i.e., pesticide registration toxicity data and open literature studies), a comprehensive 

ecological risk assessment can be performed. The EDSP Tier 2 tests complement the EDSP Tier 

1; however, results from well run Tier 2 tests provide necessary information to inform risk 

assessment. Tier 2 is the final phase of the screening and testing program and, thus, can provide 

more detailed information regarding the endocrine disruption activity of a tested substance. To 

fulfill this purpose, the MEOGRT is a longer-term study than the Tier 1 fish short-term 

reproduction (FSTRA) assay and is designed to encompass critical life stages and processes, 

cover a broad range of test concentrations, and employ a relevant route of exposure. In addition, 
Public Review Draft of December 17, 2014

Page 4 of 60

the effects associated with an EDC may be latent and not manifested until later in life or may not 

be apparent until reproductive processes occur in an organism's life history. Thus, the MEOGRT 

encompasses all the lifestages of one full generation, including effects on fertility and mating, 

embryonic development, sensitive neonatal growth and development, and transformation from 

the juvenile life stage to sexual maturity. In addition under certain circumstances, a second 

complete generation (F2) reared through reproduction may be added to the MEOGRT. The 

results from the MEOGRT should be conclusive, that is, provide a discernible cause-effect 

relationship if one exists from exposure to the test substance. 

(c) Principle of the Assay. 

This test protocol is intended to measure chemical effects on reproduction and 

reproductive development in Japanese medaka (Oryzias latipes) exposed throughout one full 

generation of their life-cycle with the potential of including a second full generation, if needed. 

The test method begins by exposing adult fish (F0 generation) to the test substance dissolved in 

water and continues through development and successful reproduction in the F1 generation. 

Thus, the MEOGRT is an extension of existing standard Fish Full Life-Cycle test protocols (Seki 

et al., 2003; Yokota et al., 2001), various OECD Test guidelines, and OCSPP 850.1500. The 

MEOGRT can also be continued through reproduction of a second generation (F2) where the 

second generation may be more sensitive (e.g., indications of transgenerational effects in other 

taxa). The F2 generation is a duplication of the F1 generation.

The MEOGRT provides data that can be used to simultaneously evaluate three general types of 

AOPs ending in reproductive impairment: a) those primarily involving disruption of the 

hypothalamus-pituitary-gonadal (HPG) endocrine axis; b) those that cause reductions in apical 

effects on survival, growth, hatch, etc. through other endocrine axes (e.g., hypothalamuspituitary-thyroid); and c) those that cause reductions in apical effects on survival, growth, hatch, 

etc. through non-endocrine mediated toxicity pathways. Some of the endocrine-relevant 

endpoints, such as the presence of anal fin papillae in medaka males, are biomarkers only 

minimally linked to adverse reproductive outcomes; whereas other apical endpoints such as 

fecundity and fertility can be directly linked to adverse population outcomes through population 

models. The non-endocrine mediated endpoints typically measured in full life-cycle (OCSPP 

Guideline 850.1500) and early life-stage (ELS; OCSPP Guideline 850.1400) chronic toxicity 

tests (e.g. survival and growth) are also included in the MEOGRT. These are used to evaluate 

the concentration-response of a chemical working through any endocrine or non-endocrine 

mediated AOP. The question of whether a test substance has endocrine-mediated effects at 

lower concentrations than non-endocrine-mediated toxicities cannot be unambiguously addressed 

by the apical endpoints alone and will rely on other diagnostic biochemical (e.g., vitellogenin 

induction), histopathological (e.g., Leydig cell hyperplasia) effects, or changes in secondary 

sexual characteristics.

(d) General Experimental Design. 

(1) Timeline. Table 1 summarizes the exposure and measurement endpoint timelines 

for the MEOGRT (see Table 2, if an additional generation is included). The standard 

MEOGRT protocol consists of continuously exposing parts of two generations of medaka 

to the test substance dissolved in treatment tank water. The F0 generation is started by 
Public Review Draft of December 17, 2014

Page 5 of 60

exposing reproductively active fish for 3 weeks, during which time the test substance 

and/or its metabolites are presumed to be distributed to the gametes and tissues of these 

fish. Reproduction is assessed in F0 fish for the first 21 continuous days of the test. At 

the beginning of the fourth week of exposure, eggs are collected to start the F1 generation 

and growth measurements are taken of the F0 fish. Fish in the F1 generation are reared 

for a total of 15 weeks post fertilization (wpf). A subset of the F1 fish is sampled at 9 

wpf for evaluation of various endpoints. Reproduction is assessed in adult F1fish for 21 

continuous days during the period of 12-14 wpf. Prior to the sacrifice of the F1 adults, 

eggs are collected and allowed to hatch, usually ending the MEOGRT. If a second 

complete generation is to be included (F2), it follows the same timeline as the F1 

generation, except hatch is not assessed after F2 reproduction.

(2) Treatments and Replication. The guideline recommends five test substance 

treatments using technical grade material and a negative control. The number of 

replicates per treatment does not remain constant throughout the MEOGRT, and the 

number of replicates in the control treatment is double of any single test substance 

treatment. In F0, each test substance treatment has six replicates while the negative 

control treatment has 12 replicates. Solvents are highly discouraged, and if used, a 

justification for both the use of a solvent and the choice of solvent should be included in 

the MEOGRT report. Also, if a solvent is used, two types of controls are necessary: a) a 

solvent control, and b) a negative control. These two control groups should each consist 

of a full complement of replicates at all points within the MEOGRT timeline. 

Throughout test organism development in the F1 generation (and F2, if included), this 

replicate structure remains the same. However, in the adult stage when F1 breeding pairs 

(also F2 breeding pairs if included) are setup, the number of reproducing pair replicates 

per treatment is optimally doubled; therefore there are up to 12 replicate pairs in each 

test substance treatment and 24 replicate pairs in the control group (and another 24 

replicate pairs in the solvent control, if needed). The determination of hatch from 

embryos spawned by the F1 pairs is done on the same replicate structure as was done for 

the embryos spawned by the F0 pairs, meaning initially six replicates per test substance 

treatment and 12 replicates in the control group(s).

(3) Endpoints. In F0, daily replicate fecundity (number of spawned eggs), and daily 

replicate fertility (number of fertile eggs) are recorded for 21 continuous days. Upon 

termination of F0, growth parameters are measured. In addition, hatching success is 

recorded for the embryos spawned by F0 fish. At the adult life-stage of F1, the primary 

data collected are related to reproduction: daily replicate fecundity (number of spawned 

eggs) for 21 continuous days, daily replicate fertility (number of fertile eggs) for 21 

continuous days, and hatching success. After this reproductive assessment, these F1 

adults (F2 adults if included) are sacrificed for growth, secondary sex characteristics, and 

histopathology assessment. At the subadult (9 wpf) life-stage of F1 (and F2 if included), 

fish are sampled for growth, secondary sex characteristics, gonad phenotype, and liver 

vitellogenin (vitellogenin 1; vtg1) mRNA assessment. Survival data are determined at 3, 

9, and 15 wpf of the F1 generation. If a F2 generation is needed, all the above data are 

collected in the same manner with the omission of hatching success. 
Public Review Draft of December 17, 2014

Page 6 of 60

(4) Genotypic Sex. Medaka have a XX/XY sex determination system in which the 

only functional gene identified on the Y chromosome is DMY (Matsuda et al. 2002). 

DMY determination for each individual fish provides a description of the genetic 

predisposition of the gender phenotype for each fish, and all endpoints are analyzed in the 

context of the genetic sex of the individual fish. Therefore, the DMY status of each fish 

within the MEOGRT is essential for proper analysis of biological data. DMY assessment 

is done on all fish just before the 9 wpf subadult sampling in order to properly setup XX 

– XY breeding pairs. In addition, the DMY information on the individual fish sampled at 

9 wpf is retained in order to properly analyze the sub-adult data.

(5) Criteria for F2 Inclusion. If circumstances warrant, a second complete 

generation (F2) could be performed. The criteria that would indicate such consideration 

for an extended study would include a high bioconcentration potential or an indication 

that transgenerational effects might be observed (e.g, transgenerational effects in other 

taxa).

Table 1. Exposure and measurement endpoint timelines for F0 and F1 generations within the 

MEOGRT.

MEOGRT Exposure and Endpoint Timeline

F0 1 2 3 4

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Lifestage Key Embryo Larvae Juvenile Subadult Adult

Endpoints

Fecundity F0 F1

 Experimental design has 7 groups of 

replicates

o 5 for test substance treatments

o 2 for control treatments (4 if solvent is used)

 Within-group design

o 12 replicates for reproduction, adult 

pathology and SSC (Wks 10 thru 18)

o 6 replicates for hatch, survival, Vtg; and - 

subadult SSC and growth (Wks 1 thru 9)

SSC: secondary sex characters; Wks: weeks; 

Vtg: vitellogenin

Fertility F0 F1

Hatch F0 F1

Survival F1 F1 F1

Growth F0 F1 F1

Vitellogenin F1

Secondary sex F1 F1

Histopathology F1

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Public Review Draft of December 17, 2014

Page 7 of 60

Table 2. Exposure and measurement endpoint timelines for F0, F1 and F2 generations within 

the MEOGRT plus F2.

MEOGRT + F2 Exposure and Endpoint Timeline

F0 1 2 3 4

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

F2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Lifestage Key Embryo Larvae Juvenile Subadult Adult

Endpoints

Fecundity F0 F1 F2

Fertility F0 F1 F2

Hatch F0 F1

Survival F1 F1 F1 F2 F2 F2

Growth F0 F1 F1 F2 F2

Vitellogenin F1 F2

Secondary sex F1 F1 F2 F2

Histopathology F1 F2

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

(e) MEOGRT Prologue.

(1) Fish. The test species of the MEOGRT is the O. latipes of the orange-red strain. 

The medaka is readily induced to breed in captivity and published methods exist for its 

culture, as well as short-term acute toxicity, early life-stage (ELS), and full life-cycle 

tests (Denny et al., 1991; Koger et al., 1999; Yokota et al., 2001; Seki et al., 2003; 

Gormley et al., 2003). All fish should be maintained on a 16-hr light:8- hr dark 

photoperiod. A record of observations and any prophylactic and therapeutic disease 

treatments during the culture period preceding the test should be maintained. The fish are 

fed live brine shrimp (Artemia) nauplii twice daily throughout the test. 

The exposure phase should be started with sexually dimorphic adult fish from a 

laboratory supply of reproductively mature animals cultured at 25 ± 1°C. The fish must 

be actively spawning prior to the start of the MEOGRT. For the group of fish used to 

initiate the test, the range in individual weights, by gender, at the start of the test should 

be kept within ± 20% of the arithmetic mean weight of the same gender. A subsample of 

fish should be weighed before the test to estimate the mean weight. The age and weight 

of the fish selected should be within 11 to 15 weeks post fertilization and >300 mg for 

females and >250 mg for males.

Test fish to be used as F0 should be selected from a single laboratory stock which has 

been acclimated for at least two weeks prior to the test under conditions of water quality 
Public Review Draft of December 17, 2014

Page 8 of 60

and illumination similar to those used in the test. In addition, each breeding pair of F0 

should be verified to be XX – XY to avoid the possible inclusion of spontaneous XX 

males. Small tissue samples of the tail fin are analyzed for DMY using the same 

procedures described during the in-life portion of the MEOGRT. A minimum of 42 

breeding pairs are considered necessary to initiate the MEOGRT (54 breeding pairs if a 

solvent control is used) to ensure adequate replication. However, it is strongly 

encouraged that several more breeding pairs than the minimum be available and 

appropriate for inclusion in the MEOGRT. In this way, pairs that for unexplained 

reasons do not spawn or stop spawning prior to the start of the test can be excluded and 

substituted with actively spawning pairs. If this recommendation is followed, the 

MEOGRT should be initiated by selecting the 42 pairs in which the daily fecundity 

values for 4 to 7 days prior to the start of the MEOGRT are closest to the mean daily 

fecundity of all available breeding pairs. In addition, the mean daily fecundity of the 

selected breeding pairs should be greater than 20 eggs/pair/day. A randomized block 

design should be used to ensure a balanced distribution of pairs to the treatments based 

on reproductive performance.

Note: this acclimation period is not an in situ pre-exposure period. It is recommended 

that test fish be obtained from an in-house culture, as shipping of adult fish is stressful 

and may interfere with reliable spawning. Fish should be fed brine shrimp twice per day 

throughout the holding period and during the exposure phase.

Mortalities in the culture fish should be recorded and the following criteria applied:

 Mortalities of greater than 10% of culture population in seven days preceding 

transfer to the test system: reject the entire batch;

 Mortalities of between 5% and 10% of population: acclimation for seven 

additional days; if more than 5% mortality during second seven days, reject 

the entire batch;

 Mortalities of less than 5% of population in seven days: accept the batch.

Fish should not receive treatment for disease in the two-week acclimation period 

preceding the test or during the exposure period. Moribund fish or fish with clinical signs 

of disease should not be used.

(2) Water. Any water in which the test species shows suitable long-term survival, 

growth, and reproduction may be used as test water. It should be of constant quality 

during the period of the test. In order to ensure that the dilution water will not unduly 

influence the test result (for example by complexation of test substance) or adversely 

affect the performance of the fish, samples should be taken at intervals for analysis. 

Measurements of heavy metals (e.g., Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations 

(e.g., Ca, Mg, Na, K, Cl, SO4), pesticides, total organic carbon and suspended solids 

should be made, for example, every six months where a dilution water is known to be 

relatively constant in quality. Some chemical characteristics of acceptable dilution water 

are listed in Table 3.
Public Review Draft of December 17, 2014

Page 9 of 60

Table 3. Some Chemical Characteristics of Acceptable Dilution Water

SUBSTANCE CONCENTRATION

Particular matter < 20 mg/L

Total organic carbon <2 mg/L

Un-ionized ammonia <1 μg/L

Residual chlorine <10 μg/L

Total organophosphorus pesticides <50 ng/L

Total organochlorine pesticides plus polychlorinated 

biphenyls

<50 ng/L

Total organic chlorine <25 ng/L

(3) Assay System. The design of the exposure system is not prescribed but certain 

requirements are essential to the proper implementation of the MEOGRT. The literature 

describes several different exposure systems used in medaka testing (Yokota et al., 2000, 

2001; Kang et al., 2002; Seki et al., 2002). A flow-through exposure system is highly 

recommend to achieve performance criteria. Static renewal systems are strongly 

discouraged. Specific details of assay system are presented in Table 4.

(i) Components. Glass or other chemically inert material should be used for 

construction of the test system. The system components including aquaria, 

dilution cells, pumps, and delivery lines, should have water-contact components 

of glass, stainless steel, Teflon®, or other inert material. However, suitable 

plastics can be utilized if it has been shown that they will not compromise the test. 

Exposure tanks should be glass aquaria with a recommended volume of 

approximately 1.8 L. 

(ii) Water Delivery. The system is required to support all chemical 

treatments and treatment controls up to 84 aquaria during reproduction (up to 108 

aquaria during reproduction if a solvent is used). The flow rate to each aquarium 

should be sufficient to maintain both biological conditions and chemical 

exposure. With 1.8 L aquaria, a flow rate of 20 mL/minute usually is sufficient. 

Delivery of each treatment to the appropriate aquaria can be accomplished with 

different systems including appropriately sized peristaltic pumps or valve-less 

metering pumps with manifold systems (see Appendix 1 for example systems). A 

flexible system will have design options that provide for a wide range of flows 

that are easily adjusted. This enables the flow to be adjusted during a study, for 

instance, if biomass reduces test agent concentrations or lowers water quality.

(iii) Lighting. Fluorescent lighting should provide a 16 hour light:8 hour dark 

photoperiod at an intensity range of 100 - 150 lux at the water surface. If 

additional light is required during certain test tasks (i.e., collection of eggs during 

reproduction), increased light intensity may be used but should be reduced back to 

the specified range as soon as possible.
Public Review Draft of December 17, 2014

Page 10 of 60

(iv) Temperature. To meet the performance criteria for the MEOGRT, 

precise temperature control is also required. One option uses low wattage silicon 

rubber heaters controlled by a digital temperature controller. This dry system 

controls tank temperatures as well as water bath systems, and has the advantage of 

allowing exposure aquaria to be removed from the system without water dripping 

and allows leaking aquaria to be easily detected. Regular recording of the 

temperature of the aquaria should be done. If a consistently high performing 

temperature regulation system is in place, a minimum of two replicates in each 

treatment should be measured daily. The replicates in each treatment should be 

rotated so that each replicate is measured approximately the same number of 

times during the MEOGRT. If temperature deviations occur, more frequent 

measurements of more than two replicates per treatment should be done to 

adequately record the temperature profile of the bioassay system. 

(v) Curtains. Medaka can react adversely (e.g, reduced fecundity) by activity 

outside their aquaria. In addition, elevated light intensity can lower fecundity. To 

maximize medaka reproduction within the MEOGRT, it is recommended that the 

test aquaria are visually isolated with curtains (or by other means) from the 

general lab environment. 

(4) Test Substance Dilution System. Due to variations in the physicochemical 

properties of test substances, different approaches for preparation of exposure water will 

be required. When possible, direct addition to water should be utilized, if the test 

substance has sufficiently high water solubility. Test substances which are liquid at room 

temperature and moderately soluble in water can be introduced using liquid:liquid 

saturator methods (Kahl et al., 1999). Test substances which are solid at room 

temperature and are moderately soluble in water can be introduced using glass wool 

column saturators (Kahl et al., 1999). Characteristics which indicate that the test 

substance may be difficult to test in aquatic systems include: high log octanol-water 

partitioning coefficients (log Kow), high volatility, susceptibility to hydrolysis, and 

susceptibility to photolysis under ambient laboratory lighting conditions. Other factors 

may also be relevant to determining testability and should be determined on a case-bycase basis. All efforts should be made to avoid solvents or carriers. If a solvent is used, 

the concentration of the solvent should not exceed 20 μL/L. All aqueous stock solutions 

should be encased in a light proof container during agitation and subsequent storage to 

prevent photo-oxidation or excessive microbial growth. The flow of the test solution 

should be verified at least weekly and the flow between replicates should not be greater 

than 10% from mean. Additionally, the system should be checked at least twice daily to 

ensure proper delivery and function.

The precise preparation of toxicant solutions to be delivered to tanks is critical to the 

success of a long-term bioassay. There are several design considerations for a toxicant 

preparation system. The preparation of the chemical treatments should be independent 

from each other to allow the adjustment of individual treatment concentrations, and the

addition or elimination of treatments. The chosen system should be capable of producing 

a large range of concentrations with a short residence time in the system allowing for 

rapid and continuous dilution and delivery to the exposure aquaria. The system should be 
Public Review Draft of December 17, 2014

Page 11 of 60

designed so that system failure does not result in an increase in the test substance 

concentration delivered to the exposure aquaria. Example dilution and delivery systems 

are shown in Appendix 1.

(5) Test Substance Dose Selection. For the purposes of this test, results from the 

Tier I EDSP studies, in particular the FSTRA (OCSPP Guideline 890.1350) should be 

used as well as any other pertinent information, to the extent possible, in determining the 

high test concentration so as to avoid concentrations that are overtly toxic. Prior to 

running the MEOGRT, a range-finding experiment should be conducted. As a 

suggestion, the rangefinder may start with newly hatched larvae and continue with an 

exposure for 48 hours. Both the number of replicates and the number of larvae per 

replicate can be kept to a minimum (i.e., 2 replicates per treatment and 10 larvae per 

replicate). The number of treatments will vary depending on the quality of the 

information regarding the test substance, but minimally there should be three doses of the 

test substance plus a control. While ancillary endpoints can be taken (i.e., growth), the 

primary data to be collected are for survival. In addition, including some reproducing 

adult pairs as another sensitive life stage with egg production as the primary endpoint is 

also recommended. The results of the range-finding experiment should serve to set the 

highest test concentration in the MEOGRT. The data from both these life-stages should 

be considered and the lower effective dose of the two studies selected as a possible high 

dose for the test. Once the highest test concentration is established, a 50% dilution series 

is recommended to establish the remaining concentrations. 

(6) Analytical Measurement of Test Substance. After preparation of the test 

substance stock solution, the concentration in the test system should be made using 

appropriate methods. Concentrations in exposure tanks should be measured prior to 

adding fish to verify that target concentrations are reached. Preceding the initiation of the 

test exposure it is important to verify that the test substance dilution and delivery system 

can provide the desired test substance concentrations to the exposure aquariums. The 

concentrations of the test substance will likely need to be measured in stock bottles (or 

saturator column elutes), mixing cells, and exposure aquaria. While these measurements 

are necessary to set up the exposure system, they will also provide insight regarding the 

aqueous stability of the test substance over the duration of the MEOGRT. Parent 

chemicals and metabolites of concern should be directly measured using methods such as 

spectrophotometry, gas chromatography-electron capture detection (GC-ECD), gas 

chromatography-mass spectroscopy (GC-MS), HPLC (high performance liquid 

chromatography) with an appropriate detector, or ion-chromatography with conductivity 

detection.
Public Review Draft of December 17, 2014

Page 12 of 60

Table 4. Summary of Test Conditions for the MEOGRT.

Parameter Information

Fish species Oryzias latipes (Japanese medaka); orange-red strain

Test type Continuous flow-through

Water temperature 25.5 ± 0.5°C 

Illumination quality Fluorescent bulbs (wide spectrum and 100-150 lux)

Photoperiod 16 h light, 8 h dark

Loading rate Embryos: 20 per incubator; Juveniles/Subadults: 12 per 

aquarium; Adults: 2 per aquarium

Example aquarium size 18x9x15 cm 

Example aquarium effective 

volume

~1.8 liters 

Volume exchanges of test 

solutions

If using example aquaria: at least 16 volume replacements/day 

(20 ml/min flow).

Age of test organisms at 

initiation

F0 ~11 weeks post-fertilization

No. of treatments 6 (5 test substance treatment plus 1 control); 7 if a solvent is 

used

No. replicates per treatment 6 per treatment for each test substance treatment; 12 for control 

treatment*; replication structure doubled for fecundity 

assessment

Aeration None unless DO reaches <60% saturation

Dilution water Previously demonstrated to be suitable for medaka

Exposure period 19 weeks total (plus 15 additional weeks if F2 included)

* If solvent is used, then 12 solvent controls as well as 12 negative controls recommended.

(f) Description of the Method.

(1) Initiation of the Test. Breeding pairs that have met the criteria detailed in 

Section (e)(1) of this guidance should be used to initiate exposure. It is recommended to 

begin with 12 replicate breeding pairs in the control treatment. There should be six 

replicate breeding pairs in each of the five test substance treatment levels for a total of 42 

breeding pairs to start the MEOGRT (54, if a solvent control is needed). While 

discouraged, if the use of a solvent has been justified, 12 negative control replicates will 

receive dilution water only and an additional solvent control group of 12 replicates will 

be added to the MEOGRT.

(2) Study Procedures.

(i) Egg Collection and Hatch. Eggs are collected during the first day (or 

first two days, if needed) of Study Week 4 for initiating the F1 generation (also in 

Study Week 18 if the F2 generation is included). After spawning, all eggs are 

collected by carefully removing any attached eggs from the female and by 

siphoning all the eggs from the bottom of each aquarium. The eggs from each 
Public Review Draft of December 17, 2014

Page 13 of 60

treatment are rolled to remove filaments, pooled; fertile, healthy eggs (embryos) 

are then randomly selected for incubation. If available, 120 embryos from each 

test substance treatment and 240 from the controls (plus an additional 240 for the 

solvent control if one is used) are placed in appropriate incubating devices 

containing 20 embryos per incubator, (see Appendix 1 for example apparatus) 

until hatch. A single incubator can be housed in each of the replicate aquaria 

along with the breeding pairs. 

It is preferable that the embryos are collected on a single day; however, if there 

are not enough embryos (120 per test substance treatment; 240 per control group), 

the embryos may be collected over two days. If collected over two days, all 

embryos within the treatments that were collected on the first day are pooled with 

those collected on the second day. Then the total pooled embryos for each 

treatment are randomly distributed to each of the replicate incubators at 20 

embryos per incubator (120 per test substance treatment; 240 per control group). 

Note: If a single treatment requires a second day of collection, all treatments 

(including controls) need to follow this procedure.

If after the second day of collection there is inadequate numbers of embryos 

within a treatment to load 20 embryos per incubator, reduce the number of 

embryos loaded within that specific treatment to 15 embryos per incubator. If 

there are not enough embryos to load 15 per incubator, reduce the number of 

replicate incubators until there are enough embryos for 15 per incubator.

The incubators must continue to receive an adequate flow of the test substance for 

the duration of the incubation period. These incubators can be located either in 

separate, dedicated aquaria or in the same aquaria containing the breeding pairs. 

If the incubators are placed in the same aquaria that contain the breeding pairs, 

care must be taken to ensure that the incubators do not become fouled with brine 

shrimp which may lead to egg/embryo mortality.

Embryos are continually agitated within the incubator, for example by constant 

aeration or delivery of test solution. Incubators should be checked daily for 

mortalities. Mortalities are recorded and all dead embryos are removed from the 

incubators. The agitation is stopped on the morning of the first expected day of 

hatch (typically at a temperature of 25.5 ± 0.5°C, this is the 8th day postfertilization), causing the embryos to settle to the bottom of the incubator which 

promotes hatching, typically over the next two days. For each treatment level on 

each day, hatchlings are counted, pooled, and systematically distributed to each of 

the replicate aquaria (Figure 1). This can be done by randomly selecting a 

hatchling from the treatment pool and sequentially adding a hatchling to the next 

replicate aquaria. Each replicate aquarium should receive 12 hatchlings. If there 

is an inadequate number of hatchlings to load at this density, reduce the number 

of replicates that are to be loaded until 12 per replicate can be achieved. 

Embryos that have not hatched after a prolonged incubation time are considered 

non-viable and discarded. This day is defined as twice the median control day of 
Public Review Draft of December 17, 2014

Page 14 of 60

hatch (at a temperature of 25.5 ± 0.5°C, typically this is the 16th day postfertilization). 

(ii) Setup of Breeding Pairs. On Study Day 78, a small tissue sample is 

taken from the caudal fin of each fish to determine the genotypic sex of the 

individual. See Appendices 3 and 4 for the detailed protocol on extracting DNA 

from caudal fins and performing the DMY analysis/genetic sex determination. 

Briefly, it is recommended that a commercially available DNA extraction kit (i.e.

DNeasy® Blood and Tissue Kit, Qiagen) be used as they provide consistently high 

quality DNA. It is essential that the integrity of the sample identification be 

maintained and that contamination between samples is prevented. The 

consequence of failure on either of these points is to have genetic sex to 

phenotypic sex mismatches that are not related to test chemical exposure, making 

the interpretation of the MEOGRT data unreliable and potentially impacting the 

acceptability of the test. Once the DNA is extracted, a polymerase chain reaction 

(PCR) method is used to amplify the DMY gene, if present, which is on the Y 

chromosome. Since the medaka has a XX/XY sex determination system, the 

presence of DMY (thus the Y chromosome) is a definitive indication of the 

individual’s genetic sex. 

This information is used to establish XX – XY breeding pairs regardless of 

external phenotype which may be altered by exposure to an EDC. As a suggested 

procedure, fish from a replicate can be housed in small cages, if possible one per 

cage, in the replicate aquarium. Alternatively, two fish can be held in each cage if 

they are distinguishable from each other. One method is to differentially cut the 

caudal fin (e.g., upper vs lower) when taking the tissue sample for DMY analysis. 

On Study Days 80 and 81 after the genotypic sex of each fish is determined, two 

XX fish and two XY fish from each replicate are randomly selected for pooling 

(Figure 1). After the 12 total XX fish (24 for control(s)) and the 12 total XY fish 

(24 for control(s)) for each treatment are pooled, and they are randomly selected 

to produce breeding pairs. If a replicate does not have either two XX or two XY 

fish, appropriate fish should be obtained from other replicates within the 

treatment. The priority is to have the recommended number of replicate breeder 

pairs in each treatment group and in the control group. 

Please note that in this process, the lineage of each replicate ends at the subadult 

sampling and is established anew with the adults. Keeping the individual fish in 

separate cages within the replicate aquarium expedites this process. It is essential 

that the fish of the breeding pairs be a) XX - XY, and b) randomly selected. 

The same sampling timepoints and processes are followed if extending the assay 

into the F2 generation. 

(iii) Sampling of Subadults. After the breeding pairs have been established, 

the fish not selected to be pooled are sacrificed for the subadult endpoints while 

maintaining the replicate structure of each treatment (Figure 1). Unlike the fish 

selected to become breeding pairs, the replicate identity of each sampled subadult 
Public Review Draft of December 17, 2014

Page 15 of 60

fish is maintained. This process is done on study day 80 and 81. It is essential 

that the fish are handled in such a way so that the genotypic sex of each fish, that 

has already been determined, can be traced back to the specific fish because all 

endpoint data is analyzed in the context of the genotypic sex of the specific fish. 

Each fish is euthanized and measured for a variety of endpoints including: 9 wpf 

survival, growth, liver vitellogenin 1 mRNA copy number, anal fin papillae 

number (secondary sexual characters (SSC)). The liver is dissected, RNA 

extracted, and the copy number of the vitellogenin I gene per ng of total mRNA is 

determined. The tail of the fish, including the anal fin, is preserved in an 

appropriate fixative or photographed so that anal fin papillae may be counted at a 

later date. It is strongly recommended that at this time another tissue sample from 

each fish sacrificed is collected and archived. This allows a post-hoc verification 

of the earlier DMY analysis in the event that genotype and phenotypic endpoints 

are non-concordant. The following appendices should be consulted for important 

guidance: Appendix 5 for the grossing procedure, Appendix 6 for counting anal 

fin papillae, and Appendix 7 and 8 for extraction of RNA and analysis of 

vitellogenin.

The same sampling timepoints and processes are followed if extending the assay 

into the F2 generation.

(iv) Assessment of Reproduction. Fecundity and fertility are assessed in 

Study Weeks 1 through 3 in the F0 generation and in Study Weeks 15 through 17 

in the F1 generation. Eggs are collected daily from each breeding pair for 21 

consecutive days. Eggs are gently removed from netted females and siphoned 

from the bottom of the aquarium each morning. Both fecundity and fertility are 

recorded daily for each replicate breeding pair. Fecundity is defined as the 

number of eggs spawned, and fertility is functionally defined as the number of 

fertile and viable eggs at the time of counting. Counting should be done as soon 

as possible after egg collection. 

For the most part, the process for the collection of eggs and hatch assessment is 

done in the same manner as described previously in this guidance. The only 

difference is in F0, there are six replicate breeding pairs per treatment (12 

replicates in controls; 24 if a solvent control is needed); whereas, in F1, eggs are 

collected from 12 replicate breeding pairs per treatment (24 replicates in controls; 

48 if a solvent control is needed). To assess embryo viability and hatch success, 

all viable embryos are pooled and systematically distributed to six replicate 

incubators (12 incubators for controls; 24 if a solvent control is needed) as was 

done in F0. If needed, a second day of collection can be used as long as the first 

and second collection days are pooled as in F0. Proceed as before, and record the 

hatch per replicate incubator so that the percent hatch can be calculated. Embryos 

that have not hatched by twice the median control day of hatch (typically the 16th

day post-fertilization) are considered non-viable and discarded. 
Public Review Draft of December 17, 2014

Page 16 of 60

The same sampling timepoints and processes are followed if extending the assay 

into the F2 generation.

(v) Sampling of Adults. The adults are sacrificed and various endpoints are 

assessed at 15 wpf. While the tail is removed and fixed or photographed to assess 

the number of anal fin papillae, no other tissue dissection is performed to maintain 

all tissues and organs in their in situ orientations. The body cavity is opened to 

allow perfusion with appropriate fixative prior to submersing the entire body in 

the fixative and each adult fish is evaluated histologically for pathology in the 

gonads, kidneys and liver tissue as described in the Histopathology Guidance 

(Appendix 10). The gonad phenotype is also derived from this evaluation. If 

needed, a tissue sample can be taken to repeat the DMY analysis to verify genetic 

sex of specific fish. For instance, it is strongly recommended that if a breeding 

pair fails to produce more than a minimal number of eggs, the genetic sex of both 

fish be re-assessed to verify that the pair was indeed XX – XY. See Appendix 11 

for detailed histology and pathology guidance.

The same sampling timepoints and processes are followed if extending the assay 

into the F2 generation.. 

(vi) Ongoing Observations. During the MEOGRT, observations of behavior 

should be made at least twice daily, and any unusual behavior should be noted. In 

addition, any mortality should be recorded and survival to 4 wpf (Study Day 44), 

from 4 wpf to 9 wpf (Study Day 80), and from 9 wpf to 15 wpf (Study Day 121) 

should be calculated. In addition, during the time leading up to the selection of 

breeding pairs, each replicate should be monitored for its first spawn. The study 

day that this occurs should be recorded, however, at this time no statistical 

analysis of this observation is suggested. 

The same sampling timepoints and processes are followed if extending the assay 

into the F2 generation.

(3) Detailed Timeline. A timeline for the MEOGRT has already been 

illustrated in Table 1 (Table 2 if the additional generation is included). The 

MEOGRT includes 4 weeks of exposure to F0 adults and 15 weeks of exposure to 

the F1 generation. The activity through the course of the MEOGRT is discussed 

below. In addition, a Microsoft® Excel spreadsheet is available in Appendix 2 

and is available as a separate electronic file that provides detailed, day-by-day 

instructions for the entire MEOGRT. It is strongly recommended that this daily 

protocol be continuously consulted before starting and during the implementation 

of the MEOGRT as it details the activities performed during each day of the test. 

An additional F2 generation can be included, if necessary, that would go through 

reproduction ending at 15 wpf. The F2 generation is identical to the F1 

generation, and likewise overlaps the previous generation by one week; therefore, 

the inclusion of a F2 generation adds 14 weeks to the MEOGRT timeline. 
Public Review Draft of December 17, 2014

Page 17 of 60

Study Weeks 1-3. The F0 generation spawning fish, that have met the 

selection criteria detailed above, are exposed for three weeks to allow the 

developing gametes and gonadal tissues to be exposed to the test substance. Each 

replicate aquarium houses a single XX – XY breeding pair of fish for a test total 

of 42 breeding pairs (54 breeding pairs if a solvent control is needed). Spawned 

eggs are collected daily for 21 consecutive days starting at Study Day 1, counted, 

and assessed for fertility. 

Study Week 4: On Study Day 22, eggs are collected from each aquarium 

and each female, pooled by treatment, and systematically distributed to suitable 

incubation vessels (6 per treatment and 12 for controls) as detailed in (f)-(2)-(iv). 

Again, the vessels may be placed in separate “incubator aquaria” set up for each 

treatment or in the replicate aquarium that upon hatch will contain the hatched 

larval fish. Ideally, all embryos used to start F1 should be collected on the same 

day; however, a second day of collection may be necessary if an insufficient 

number of viable embryos are collected on the first day. If a second day of 

collection (Study Day 23) is needed, all embryos from both days should be pooled 

and then systematically redistributed to each of the treatment replicates.

On Study Day 24, the F0 breeding pairs are terminated; weight and length are 

recorded. 

It is important for successful hatch, primarily by minimizing mortality from 

pathogens and fungal infestations, to agitate the embryos in the incubation 

vessels. Aeration is a convenient method to properly agitate the eggs, and 

example apparatus are shown in Appendix 1. 

Study Weeks 5-6. On Study Day 30, the agitation of the incubating 

embryos is stopped to expedite hatching. As embryos hatch on each day, 

hatchlings are pooled by treatment and then systematically distributed to each 

replicate aquarium within a specific treatment with no more than 12 hatchlings 

placed into each aquarium. This is done by randomly selecting hatchlings and 

placing a single hatchling in successive replicates in an indiscriminate draw, 

moving in order through the specific treatment replicates until all replicates within 

the treatment have 12 hatchlings. If there are not enough hatchlings to fill all 

replicates then ensure as many replicates as possible have 12 hatchlings to start 

the F1 phase of the MEOGRT. The appropriate data are collected to calculate the 

hatching success of each replicate incubator. As detailed previously, embryos 

that have not hatched by twice the median control day of hatch (typically the 16th

day post-fertilization) are considered non-viable and discarded. 

Study Weeks 6-11. On Study Day 44, survival of the larval fish to this 

point is recorded as the number per replicate out of the initial number of 

hatchlings, nominally 12. The exposure continues as the larval fish develop into 

subadults. 
Public Review Draft of December 17, 2014

Page 18 of 60

Study Week 12. On Study Day 78, a small tissue sample is taken from 

the caudal fin of each fish to determine the genotypic sex of the individual by 

DMY analysis. This information is used to establish XX - XY breeding pairs. 

The DMY data for all the remaining subadult samples is retained to ensure that all 

endpoint data can be related to the genetic sex of each individual fish. On Study 

Days 80 and 81, after the genotypic sex of each fish is determined, 12 breeding 

pairs per treatment (24 breeding pairs for controls) are randomly established as 

detailed in (f)-(2)-(ii) while the 8 (nominal) remaining fish per replicate are 

sampled for the various subadult endpoints as described in (f)-(2)-(vi). It is 

essential that the DMY status and therefore the genotypic sex (XX or XY) of 

every fish be determined at this time. See Appendices 3 and 4 for detailed 

protocols on DNA extraction and DMY analysis. 

Study Weeks 13-14. The exposure continues as the subadult fish selected 

to be breeding pairs develop into adults.

Study Weeks 15-17. On Study Day 98, eggs are removed from both the 

aquaria and the females to guarantee an accurate enumeration of egg numbers 

spawned the next day. Spawned eggs are collected daily for 21 consecutive days 

from Study Days 99-119, counted, and assessed for fertility. 

Study Week 18. On Study Day 120, embryos are collected, pooled from 

each treatment, and systematically distributed to suitable incubation vessels (6 per 

treatment and 12 for controls; plus an additional 12 controls if a solvent control is 

used) with no more than 20 embryos per replicate (120 eggs for each test 

substance treatment and 240 eggs for the controls; plus an additional 240 for the 

solvent control if one is used) allowed to incubate. If a second day of embryo 

collection (Study Day 121) is needed, all embryos from the first day (Study Day 

120) should be pooled with the embryos from the second day, and then 

systematically redistributed to each of the treatment replicates. This procedure is 

detailed in (f)-(2)-(iv). After egg collection, the F1 breeding pairs are terminated 

and analyzed for the various adult endpoints as described in (f)-(2)-(vi).

Study Week 19. On the morning of the first expected day of hatch 

(typically this is the 8th day post-fertilization), approximately Study Day 128, the 

agitation of the incubating embryos is stopped to expedite hatching. Incubators 

are evaluated daily for hatchlings. Each day the hatchlings are tallied and 

discarded. Embryos that have not hatched after a prolonged incubation time are 

considered non-viable and discarded. This day is defined as twice the median 

control day of hatch (typically the 16th day post-fertilization). The total number 

of hatchlings in each replicate is recorded to determine hatch success.

Optional F2 Generation. The optional F2 generation is identical to the 

F1 generation with a minor difference at the beginning of F2. The F1 generation 

is started with the pooled embryo output from six replicates in each treatment and 

12 replicates in the control(s). Since the reproductive assessment is done on 

double the number of replicates, the F2 generation is started from the pooled 
Public Review Draft of December 17, 2014

Page 19 of 60

embryo output from 12 replicates in each treatment and 24 replicates in the 

control(s). In all other aspects, the F2 generation is identical to the F1 generation.

Endpoints

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

20

eggs

20

eggs

20

eggs

20

eggs

20

eggs

20

eggs

12

XX

12

XY

pool 

viable eggs

distribute

pool 

hatchlings

distribute

12

fish

12

fish

12

fish

12

fish

12

fish

12

fish

DMY

assessed

subadult

samples

DMY

assessed

subadult

samples

subadult

samples

subadult

samples

subadult

samples

subadult

samples

DMY

assessed

DMY

assessed

DMY

assessed

DMY

assessed

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

XX

XY

pool 

viable eggs

distribute

hatch survival growth, VTG, SSC reproduction, pathology, SSC hatch

F0F1

20

eggs

20

eggs

20

eggs

20

eggs

20

eggs

20

eggs

Figure 1. Pooling strategy used during the MEOGRT. The figure represents one treatment or 1⁄2 of a 

control. If the F2 generation is maintained through reproduction (not shown), it is identical to the F1 

generation. Notice that at each pooling, the replicate lineage is re-established so that a specific replicate 

identity is not continuous through time. 

(3) Feeding Schedule. The following feeding schedule is recommended to ensure 

adequate growth and development to support robust reproduction. Deviations from this 

feeding schedule may be acceptable, but they should be tested to verify that acceptable 

growth and reproduction are observed. In order to follow the suggested feeding schedule, 

the dry weight of brine shrimp per volume of brine shrimp slurry needs to be determined 

prior to starting the MEOGRT. This can be done by weighing a defined volume of brine 

shrimp slurry that has been dried for 24 hours at 60°C on pre-weighed pans. To account 

for the weight of the salts in the slurry, an identical volume of the same salt solution used 

in the slurry must also be dried, weighed, and subtracted from the dried brine shrimp 

slurry weight. Alternatively, the brine shrimp can be filtered and rinsed with distilled 

water before drying. Thereby eliminating the need to measure the weight of a “salt 

blank”. This information is used to convert the information in Table 4 from dry weight 

of brine shrimp to volume of brine shrimp slurry to be fed per fish. In addition, weekly 
Public Review Draft of December 17, 2014

Page 20 of 60

aliquots of the brine shrimp slurry should be weighed to verify the correct dry weight of 

brine shrimp is being fed. 

Table 4. Feeding Schedule.

Time

(post-hatch)

Brine Shrimp

(mg dry weight/fish/day)

Time

(post-hatch)

Brine Shrimp

(mg dry weight/fish/day)

Day 1 0.5 Day 12 4.2

Day 2 0.5 Day 13 4.5

Day 3 0.6 Day 14 4.8

Day 4 0.7 Day 15 5.2

Day 5 0.8 Day 16-21 5.6

Day 6 1.0 Week 4 7.7

Day 7 1.3 Week 5 9.0

Day 8 1.7 Week 6 11.0

Day 9 2.2 Week 7 13.5

Day 10 2.8 Week 8-sacrifice 22.5

Day 11 3.5

(4) Analytical Chemistry Schedule. After the exposure is initiated, it is important to 

monitor the concentration of the test substance in the exposure aquariums frequently 

enough to allow accurate characterization of the treatment concentrations, and to 

determine whether the dilution/exposure system is performing as desired. If it is not, then 

the appropriate adjustments can be made to mitigate excursions of the test substance 

concentrations in each treatment as necessary. Weekly sampling of at least two replicate 

aquaria from each treatment is usually sufficient to define the concentration of each 

treatment through time. One of the samples should be taken from the same treatment 

replicate at each sampling time and the other sample should be selected systematically 

from one the other possible treatment replicates. This sampling strategy will provide 

estimates of test substance concentration in each treatment through time as well as 

estimates of the within-treatment concentration variance. It is also important to evaluate 

other possible sources of variance in the analytical chemistry process by using 

appropriate measurement replication and chemical standards. For test substances that 

appear to be unstable or are subject to biological degradation in the dilution/exposure 

system, it may be necessary to measure treatments more than once per week. 

(5) System Cleaning Schedule. The chemical dilution and exposure systems used 

for the MEOGRT are in contact with water and diluted chemical for a relatively long 

period. Biofilms invariably form on most wetted surfaces. The microbiological 

community that establishes itself will often be able to metabolize the compound being 

assessed. Often, detectable decreases in test substance concentration can be observed 

within three weeks of the start of the assay. Routine cleaning and disinfection of all 

wetted components in the diluter/delivery system, and exposure aquaria should be 

performed at least every three weeks.
Public Review Draft of December 17, 2014

Page 21 of 60

A suggested cleaning protocol would be to divert the exposure delivery lines from each 

replicate aquarium. The replicate aquaria can usually be left static without flow for the 

necessary time. The system components that can be removed should be cleaned with 

detergent and scrubbed with an abrasive pad. The delivery lines should be sanitized 

using a cold sterilant product such as Minncare® Cold Sterilant, (a solution of 22% 

hydrogen peroxide and 4.5% peroxyacetic acid). As an example, the cold sterilant 

solution should be mixed with water to a solution of 50X (2% concentration), and used to 

fill the toxicant delivery line circuit. Allow the lines to soak without flow for a minimum 

of 20 minutes. Rinse the delivery lines immediately with diluent water and check for 

residuals using Minncare® Residual Test Strips. Flush the lines until the cold sterilant 

concentration is below detection. Reassemble the system and return the delivery lines to 

the exposure aquaria.

As a precautionary note, diluter systems are designed to the particular needs of each user. 

The materials of construction may not be compatible with a specific sterilant. Before 

disinfecting any dilution system, an evaluation as to whether the system is compatible 

with the sterilant solution used needs to be done. Analysis of chemical concentrations 

should be performed both before and after any cleaning of the exposure system. 

(6) Environmental Conditions. Water quality characteristics should be measured 

regularly across replicates and treatments to document adequate water quality to sustain 

healthy medaka. Dissolved oxygen, and pH should be measured in two replicates in each 

treatment at least once a week. Temperature should be monitored continuously and 

recorded daily. The measured replicates should be rotated within each treatment so that 

all replicates are measured roughly the same number of times over the MEOGRT. 

Ammonia should be measured monthly in each treatment. The frequency of 

measurement should increase if the specific water quality parameter is approaching 

biologically significant levels: dissolved oxygen ≤5.0 mg/L, and un-ionized ammonia > 

35 μg/L. The dilution water alkalinity, hardness, and total organic carbon should be 

documented at least once during the MEOGRT.

(7) Randomization. At all stages and in most, if not all tasks, it is important to 

include randomization. This includes, but is not necessarily limited to, the following 

procedures. 

 The order in which food is delivered to the aquaria should be randomized to 

mitigate potential changes in food quantity or quality during the course of 

feeding. 

 It is essential to randomize the selection of fish when distributing them into the 

replicates as hatchlings, and designating them to either become part of breeding 

pairs or be sampled as subadults.

 When fish are sacrificed either as adults or subadults, the order in which the 

replicate aquaria are sampled should be random.
Public Review Draft of December 17, 2014

Page 22 of 60

(g) Performance Criteria. 

Failure to meet a single performance criterion, while a warning sign, would in general not 

be expected to compromise the performance of the entire MEOGRT. However, failure in several 

criteria or failure to meet the fecundity criterion could result in the rejection of the test. The 

following performance criteria are generally expected for an acceptable test:

 The dissolved oxygen concentration should be between 60% and 100% of the air 

saturation value throughout the test;

 The recommended mean temperature throughout the test is 25.5°C ± 0.5°C. Brief 

excursion from the mean by individual aquaria should not be more than 2°C. 

Replicates within a treatment should not be statistically different from each other, 

and treatments within the test should not be statistically different from each other.

 Greater than 80% of control embryos should hatch. There should be at least 80% 

survival of control fish after hatching through 21-d post-hatch and greater than 

90% survival from 3 wpf to 8 wpf. Mortalities due to technical errors (handling) 

should not be included in these analyses. 

 Control XX and XY fish should have a mean weight of at least 100 mg and a 

mean length of at least 20 mm at the subadult sampling. Growth of substantially 

less than these values suggests the fish will not achieve sufficient size to spawn 

with acceptable fecundity. 

 XX males are occasionally observed in cultured medaka populations. Because of 

practical limitations, this phenomenon may be impossible to eliminate; however, 

efforts should be made to select against XX males in breeding cultures. The 

prevalence of XX males (determined by comparison of genotype and histological 

sex) in the control population should be less than 5%. In addition, less than 2% of 

control XY fish should have intersex of any severity in the adult histopathology 

samples. 

 Control fecundity should be greater than 20 eggs per pair per day. These should 

be greater than 75% fertile. For adequate statistical power, 16 pairs in the control 

group should exceed these minimum requirements.

 General toxicity as measured by liver pathology should be low in the adult 

histopathology samples.

 There needs to be at least partial survival of post-hatch fish in the second highest 

treatment (T5) in the F1 generation.

 Although decreased reproduction may be observed in the higher treatments there 

should be adequate reproduction in at least the third highest treatment (T4) and all 

lower treatments of F0 to fill the hatching incubators. Furthermore, there should 
Public Review Draft of December 17, 2014

Page 23 of 60

be adequate embryo survival in T4 and lower treatments in F1 to allow endpoint 

evaluation at the subadult sampling. Additionally, there should be at least 

minimal survival (~20%) in T5 of F1.

(h) Data Analysis.

(1) Statistical Analysis. The types of biological data generated in the MEOGRT are 

not unique to it and except for pathology data, many appropriate statistical methodologies 

have been developed to properly analyze similar data depending on the characteristics of 

the data including normality, variance homogeneity, whether the study design lends itself 

to hypothesis testing or regression analysis, parametric versus non-parametric tests, etc. 

In general principle, the suggested statistical analyses follow the recommendations of the 

OECD for ecotoxicity data (OECD 2006) and can be seen in Figure 2

It is assumed that most often the datasets will display monotonic responses. 

Additionally, the issue of using a one-tailed statistical test versus a two-tailed statistical 

test should be considered. Unless there is a biological reasoning that would make a onetailed test inappropriate, it is suggested that one-tailed tests be used. While the following 

section recommends certain statistical tests, if more appropriate and/or powerful 

statistical methods are developed for application to the specific data generated in the 

MEOGRT, those statistical tests would be used to leverage those advantages. 

Since DMY information was determined for all test fish, the data should be analyzed for 

each genotypic sex separately. Failure to do this will greatly reduce the statistical power 

of any analysis. 

(i) Histopathology data. For histopathology data which are in the form of 

severity scores, a new statistical test termed RSCABS (Rao-Scott CochranArmitage by Slices) has been developed (Green et al. 2014). RSCABS uses a 

step-down Rao-Scott adjusted Cochran-Armitage trend test on each level of 

severity in a histopathology response. The Rao-Scott adjustment incorporates the 

replicate vessel experimental design into the test. The by-slices procedure 

incorporates the biological expectation that severity of effect tends to increase 

with increasing doses or concentrations, while retaining the individual subject 

scores and revealing the severity of any effect found. The RSCABS procedure 

not only determines which treatments are statistically different (i.e., have higher 

prevalence of pathology than controls), but it also determines at which severity 

score the difference occurs providing much needed context to the analysis.

(ii) Time to event data. Within the recommended MEOGRT protocol, the 

observation of the first spawn within each replicate just before determining 

breeding pairs is the only information that could be analyzed as a time to an event. 

However, the recommendation is simply to record this observation for each 

replicate, include it as an observation in the study report, but not to analyze it with 

a formal statistical procedure.
Public Review Draft of December 17, 2014

Page 24 of 60

(iii) Fecundity data. The raw data are recorded and presented in the study 

report as the fecundity (number of eggs) per replicate for each day. The replicate 

mean of the raw data should be calculated then a square root transformation 

applied. A one-way ANOVA on the transformed replicate means should be 

calculated followed by Dunnett contrasts. 

(iii) All other biological data. The first formal check of the data is to test 

whether it violates the assumptions of monotonicity by using linear and quadratic 

contrasts. If the quadratic contrast is significant and the linear contrast is not, the 

data are considered as having failed the test for monotonicity. The underlying 

assumption, given proper dose selection, is that the data will be monotonic. If the 

data are monotonic, a Jonckheere-Terpstra on replicate medians trend test (as 

advised in OECD 2006) is recommended. 

If the data are non-monotonic, in particular because of the reduced response of the 

highest one or two treatments, consideration should be given to censoring the 

dataset so that the analysis is done without those treatments. This decision will 

need to be made with professional judgment and all available data, especially data 

that indicates overt toxicity at those treatment levels. 

For weight and length, no transforms are recommended. However, a log 

transformation is recommended for the vitellogenin data; a square root 

transformation is recommended for the SSC data (anal fin papillae); an arcsinesquare root transformation is recommended for the data on percent hatch, percent 

survival, and percent fertile eggs. 

The biological data from adult samples has one measurement per replicate, that is, 

there are one XX fish and one XY fish per replicate aquarium. Therefore, it is 

recommended that a one-way ANOVA be done on the replicate means. If the 

assumptions of the ANOVA (normality and variance homogeneity as assessed on 

the residuals of the ANOVA by Shapiro-Wilks test and Levene’s test, 

respectively) are met, Dunnett contrasts should be used to determine treatments 

that were different from the control. On the other hand, if the assumptions of the 

ANOVA are not met, then a Dunn’s test should be done to determine which 

treatments were different than control. A similar procedure is recommended for 

data that is in the form of percentages (fertility, hatch, and survival). 

The biological data from subadult samples has from 1 to 8 measurements per 

replicate, that is, there can be variable number of individuals that contribute to the 

replicate mean for each genotypic sex. Therefore, it is recommended that a mixed 

effects ANOVA model be used followed by Dunnett contrasts, if the normality 

and variance homogeneity assumptions were met (on the residuals of the mixed 

effects ANOVA). If they were not met, then a Dunn’s test should be done to 

determine which treatments were different than control.
Public Review Draft of December 17, 2014

Page 25 of 60

Figure 2. Flow chart for the recommended statistical procedures for MEOGRT data analysis. 

(2) Special Data Analysis Considerations.

(i) Use of Compromised Treatment Levels. Several factors are considered 

when determining whether a replicate or entire treatment demonstrates overt 

toxicity and should be removed from analysis. Overt toxicity is defined as >4 

mortalities in any replicate between 3 wpf and 9 wpf that can only be explained 

by toxicity rather than technical error. Other signs of overt toxicity include 

hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and any 

clinical signs of disease. For sub-lethal signs of toxicity such as reduced growth 

and pathology of non-gonadal tissue, qualitative evaluations may be necessary, 

and should always be made in reference to the appropriate control group. If overt 

toxicity is evident in the highest treatment(s), it is recommended that those 

treatments be censored from the analysis.

(ii) Solvent controls. The use of a solvent should only be considered as a last 

resort, when all other chemical delivery options have been considered. If a 

solvent is used, a justification for both the use of a solvent and the choice of 

solvent should be included in the MEOGRT report. A full complement of 

replicates for both a negative control (dilution water only) and a solvent control 

(solvent plus dilution water) should be included in the MEOGRT. At the 

termination of the test, an evaluation of the potential effects of the solvent must be 

performed. This is done through a statistical comparison of the solvent control 
Public Review Draft of December 17, 2014

Page 26 of 60

and the negative control. If statistically significant differences are detected 

between the solvent control and negative control groups, then best professional 

judgment should be used to determine if the validity of the test is compromised. 

If no statistical difference is present between the two control groups, the 

treatments exposed to the test agent should be compared to the solvent control.

(i) Data Reporting. 

(1) Test report. The test report should, at minimum, include the following 

information:

(2) Test substance. Physical nature and relevant physical-chemical properties, 

chemical identification data including purity and analytical method(s) for quantification 

of the test substance where appropriate, source, CAS number, lot number should be 

reported.

(3) Test animals. At a minimum, the supplier, available culture history including 

specific strain information, and any pretreatment should be reported.

(4) Test conditions. Test procedure used (test type, loading rate, stocking density, 

etc.); method of preparation of stock solutions and flow-rate; nominal test substance 

concentrations, means of the measured values and standard deviations in test tanks and 

method by which these were attained and evidence that the measurements refer to the 

concentrations of test substance in true solution; dilution water characteristics (including 

pH, hardness, alkalinity, temperature, dissolved oxygen concentration, residual chlorine 

levels, total organic carbon, suspended solids, and any other measurements made); water 

quality within test tanks: pH, hardness, temperature, and dissolved oxygen concentration; 

detailed information on feeding (e.g. brine shrimp source, analyses for relevant 

contaminants if necessary, e.g., PCBs, PAHs and organochlorine pesticides, any 

deviations from the feeding protocol and rationale for those deviations), source and 

treatment of dilution water, average and ranges of water chemistry parameters, light 

intensity, and tank dimensions.

(5) Results. Evidence that controls met the performance criteria; analytical 

techniques used, statistics, treatment of data and justification of techniques used; 

tabulated data preferably using the suggested data template for MEOGRT (Appendix 10); 

detailed report on histopathology including tabulated data using suggested pathology 

spreadsheet (Appendix 10); results of the statistical analysis preferably in tabular and 

graphical form; incidence of any unusual reactions by the fish and any visible effects 

produced by the test substance; mean, standard deviation, and range for each test 

endpoint. All data should be collected using electronic or manual systems which 

conform to good laboratory practices (GLP).

(j) Interpretation of Results. 

The MEOGRT as presented is intended to serve as a definitive Tier 2 test within the 

EDSP. As such, the goal of the MEOGRT is to determine whether adverse effects associated 
Public Review Draft of December 17, 2014

Page 27 of 60

with putative endocrine-mediated pathways of a test substance occur in medaka, and to 

quantitatively evaluate those apical effects. In addition, the MEOGRT must include exposure 

during the most sensitive life stages, and provide the opportunity for identification of doseresponse effects. As a Tier 2 test, the MEOGRT should complement the Tier 1 battery. 

Because the statistical analysis methods recommended tend to favor detection of monotonic 

responses, it is important to consider any significant finding an indication of an adverse response. 

Also, the suite of endpoints included is deemed necessary to provide information both on 

important apical endpoints that are relevant at the population level, and on endpoints that 

characterize the AOPs that might be affected by exposure to the test agent. 

The test should define a NOEC and have at least one concentration without a statistically 

significant effect on any of the observed endpoints.

It is important to note, however, that if a given exposure level results in substantial mortality or 

other overt signs of toxicity, responses in other endpoints may be due to general toxicity, and not 

necessarily mediated via a primary interaction with the endocrine system. Any lower treatment 

level(s) should be examined for effects outside of the range of general toxicity. If all test 

substance concentrations exhibit mortality or effects on apical endpoint such as growth, 

fecundity, sex reversal, and intersex, then the assay would need to be repeated with lower 

concentrations before inferences about possible endocrine activity could be made.

(k) References.

(1) Denny J.S., Spehar R.L., Mead K.E., Yousuff S.C. 1991. Guidelines for culturing 

the Japanese Medaka, Oryzias latipes. US EPA/600/3-91/064.

(2) Gormley K., Teather K. 2003. Developmental, behavioral, and reproductive effects 

experienced by Japanese medaka in response to short-term exposure to endosulfan. 

Ecotox Environ Safety 54:330-338.

(3) Green J.W., Springer T.A., Saulnier A.N., Swintek J. 2014. Statistical analysis of 

histopathology endpoints. Environ Toxicol Chem 33:1108-1116.

(4) Kahl M.D., Russom C.L., DeFoe D.L., Hammermeister D.E. 1999. Saturation units 

for use in aquatic bioassays. Chemosphere 39:539-551.

(5) Kang I.J., Yokota H., Oshima Y., Tsuruda Y., Oe T., Imada N., Tadokoro H., Honjo 

T. 2002. Effects of Bisphenol A on the reproduction of Japanese Medaka (Oryzias 

latipes). Environ Toxicol Chem 21:2394-2400.

(6) Koger C.S., Teh S.J., Hinton D.E. 1999. Variations of light and temperature 

regimes and resulting effects on reproductive parameters in Medaka (Oryzias 

latipes). Biology of Reproduction 61:1287-1293.

(7) Matsuda M., Nagahama Y., Shinomiya A., Sato T., Matsuda C., Kobayashi T., 

Morrey C., Shibata N., Asakawa S., Shimizu N., Hori H., Hamaguchi S., Sakaizumi 
Public Review Draft of December 17, 2014

Page 28 of 60

M. 2002. DMY is a y-specific dm-domain gene required for male development in 

the medaka fish. Nature 417:559-563.

(8) OECD 2006. Current approaches in the statistical analysis of ecotoxicity data: a 

guidance to application. Environmental Health and Safety Publications. Series on 

Testing and Assessment, No. 54. Paris, France.

(9) Seki M., Yokota H., Matsubara H., Tsuruda Y., Maeda M., Tadokoro H., 

Kobayashi K. 2002. Effect of ethinylestradiol on the reproduction and induction of 

vitellogenin and testis-ova in medaka (Oryzias latipes). Environ Toxicol Chem

21:1692-1698.

(10) Seki M., Yokota H., Matsubara H., Maeda M., Tadokoro H., Kobayashi K. 2003. 

Fish full life-cycle testing for the weak estrogen 4-tert-pentylphenol on medaka 

(Oryzias latipes). Environ Toxicol Chem 22:1487-1496.

(11) Yokota H., Tsuruda Y., Maeda M., Oshima Y., Tadokoro H., Nakazono A., Honjo 

T., Kobayashi K. 2000. Effect of bisphenol A on the early life stage in Japanese 

medaka (Oryzias latipes). Environ Toxicol Chem 19:1925-1930.

(12) Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H, Honjo T, Kobayashi K. 

2001. Life-cycle toxicity of 4-nonylphenol to medaka (Oryzias latipes). Environ 

Toxicol Chem 20:2552-2560.
Public Review Draft of December 17, 2014

Page 29 of 60

(j). List of MEOGRT Appendices. Page

(1) Example Infrastructure............................................................. 30

(2) Example Daily Calendar.......................................................... 36

(3) DNA Extraction from Fin Clips................................................ 40

(4) DMY Analysis of DNA........................................................... 41

(5) Subadult Sampling – Grossing................................................... 46

(6) Counting Anal Fin Papillae..................................................... 48

(7) RNA Extraction from Medaka Liver............................................ 49

(8) Preparation of RNA for Standard Curve Used during VTG1 QPCR....... 50

(9) QPCR to Quantify Vtg1 mRNA in Liver....................................... 55

(10) Data Reporting Templates....................................................... 59

(11) Histopathology Guidance........................................................ 61
Page 30 of 60

APPENDIX 1: Example Infrastructure

1. Background. 

This appendix illustrates examples of infrastructural components that have been successfully 

used to conduct this test protocol. 

2. Egg Incubators. 

The smooth transition from the adults of a generation to the fry of the next generation is critical 

to the success of the assay. The embryos require careful handling during this period. The design 

of an embryo incubator needs to address both husbandry and exposure concerns. Careless 

embryo husbandry can result in mortalities and lost embryos resulting in either insufficient fry to 

continue to the next generation or artificially low numbers for hatch success. Furthermore, 

embryo development is an important exposure window in which the treatment chemical 

concentration must remain stable. Therefore the incubator design should allow for both 

continuous renewal of the test chemical and easy access to collect water samples from inside the 

incubator in order to determine the chemical concentration within the incubator. Easy and clean 

removal of the incubator from the exposure system is also a requirement in order to observe the 

embryos under magnification, to remove non-viable embryos and to remove hatched fry. 

Elements critical to embryo hatching success are: monitoring of the embryos and immediately 

removing ones that are non-viable; precision handling of embryos during transfers between 

containers; and precise control of air and/or water flow in the incubator. 

The following are short descriptions and illustrations of incubators that have been used to 

successfully hatch medaka embryos.

2.1 Flow Though Incubator Tube.

(a) Pyrex No. 8240-100 tube

(b) Tank

Figure 1. Flow Though Incubator Tube. (a) This type of incubator is constructed from a 

Pyrex No. 8240-100 tube, cut to a length of 14 cm with slots cut for overflow. (b) A stainless 

steel screen is placed over the slots, covering the slots and preventing the eggs from washing out 

the slots while still allowing for overflow. The tank’s influent delivery line discharges directly 

into the incubator. A disposable pipette bubbles air to gently agitate the embryos. 
Public Review Draft of December 17, 2014

Page 31 of 60

2.2 Threaded Cap Incubator. 

Figure 2. Threaded Cap Incubator. This incubator consists of a transected glass 

centrifuge tube, connected by a stainless steel sleeve and held in place by the centrifuge 

screw top cap. A small glass or stainless steel tube projects through the cap and is 

positioned near the rounded bottom, gently bubbling air to suspend the eggs and reducing 

between-egg transmission of saprophytic fungal infections while also facilitating 

chemical exchange between the incubator and the holding tank. 

2.3 Syringe Style Incubator. 

Figure 3. Syringe Style Incubator. This incubator consists of a fluid dispensing 

syringe assembly containing stainless steel screens located at the top and bottom of the 

syringe, ensuring that the eggs are contained within the syringe body. A series of valves 

allow the flow to the incubator to be precisely regulated and/or diverted to the aquaria or 

to drain as needed. The flow of fluid through the incubator should be sufficient to provide 

agitation to the eggs.
Public Review Draft of December 17, 2014

Page 32 of 60

3. Isolation Chambers (Figure 4). 

From Study Week 6 through Study Week 11 of the F1 generation, twelve fish are held in each 

replicate tank. The genotypic sex of each fish is determined during Study Week 12. Each fish 

will have a small sample of the caudal fin taken to determine its sex. The DMY analysis has a 

turnaround time of 24-48 hours. The fish need to be held as identifiable individuals until their 

genetic sex has been determined and they can be assigned to a breeding pair or sacrificed for the 

subadult endpoints. Isolation chambers are used to hold the fish, with two fish contained in each 

chamber. The caudal fin of each fish is differentially cut with one a dorsal and the other 

receiving a ventral cut. Six chambers are set up in each aquarium. The placement of the 

chamber in the replicate aquarium is pre-determined to maintain the identity of each fish. 

Figure 4. Isolation Chamber Illustration. On the left, a single chamber. On the right, aquarium 

with six chambers.

4. Temperature Control and Monitoring. 

Fecundity and growth endpoints require precise temperature control and monitoring. A dry 

system using low wattage rubber silicon heaters controlled by a digital temperature controller is 

recommended (Figure 5). The heating system should be sized to avoid large temperature swings 

if a component fails. To further aid in temperature control, the aquaria should receive treatment 

water that is temperature controlled at the nominal test temperature. The tank heater system only 

inputs heat to compensate for ambient loss. It is also recommended that the heating system is 

linked to a data collection system that provides real time temperature monitoring and data 

logging for each aquaria.
Public Review Draft of December 17, 2014

Page 33 of 60

Figure 5. Tank temperature and monitoring display.

5. Delivery Systems. 

The flow of water to each tank should be uniform to ensure that the water quality is comparable 

in all treatments. Requirements of such a system are:

 The system should provide reliable and consistent flow. It should be easily 

adjusted to a wide range of flow, allowing it to be adjusted in response to changes 

in the biomass, water quality or chemical degradation.

 Ease of maintenance should be considered in the design of a water delivery 

system. 
Public Review Draft of December 17, 2014

Page 34 of 60

(1) Multi-channel Peristaltic Pump (Figure 6). 

Figure 6. Multi-channel Peristaltic Pump. One system that meets these requirements is a 

multi-channel peristaltic pump system which can deliver treatment water from the treatment 

reservoir to each replicate exposure aquarium. These systems can reliably deliver flows up to 50 

ml/min/channel. Delivery lines should be sterilized on a set maintenance schedule to control inline biological growth and the peristaltic tubes should be replaced on a regular basis since the 

flow will diminish as the tubing wears.

(2) Positive Displacement Pump and Manifold (Figure 7).

Figure 7. Positive Displacement Pump and Manifold. Another type of system that meets the 

above requirements is a positive displacement dilution pump pressurizing a manifold that has a 

series of programmable solenoids directing flow to the replicate exposure aquaria. This system 

eliminates the treatment reservoir and peristaltic pump. The treatment flow is controlled by the 

sped of the pump which continuously prepares treatment solution. Treatment solution is 

sequentially dispensed to the replicate tanks by the opening of a solenoid directing flow to a tank.
Public Review Draft of December 17, 2014

Page 35 of 60

6. Chemical Dilution System. 

The precise preparation of toxicant solutions to be delivered to exposure tanks is critical to the 

success of a long-term bioassay. The design considerations for a toxicant preparation system 

are:

 The preparation of treatments should be independent. This enhances study design 

flexibility by allowing the adjustment of individual treatment concentrations, the addition 

or elimination of treatments, and the selection of concentration gradients based on the 

experimental design and not on system limitations.

 The ability to produce a large range of treatment concentrations.

 The toxicant should have short residence time in the system. The toxicant should 

undergo continuous dilution with rapid delivery to the exposure tanks. 

 Excessive chemical should not be sent to waste.

 If the system fails the toxicant levels should drop.

One toxicant preparation system meeting these requirements incorporates dual headed positive 

displacement pumps which have independent pump heads linked by a common drive motor. 

Each treatment solution is prepared by a dedicated pump (Figure 8). The pump links a flow of 

dilution water to a flow of toxicant which are combined at a T fitting and directed through a 

static mixer into the treatment reservoir. The ratio of dilution water to toxicant is set by 

adjusting the piston displacement volume of the pump heads. The piston adjustment is 

independent for each pump head and flow ratios of 1:1 to 200:1 can be set up. This range can be 

expanded by swapping in higher or lower flow pump heads. The flow to the treatment reservoir 

is controlled by the speed of the drive motor. The pump speed can be manually adjusted or 

controlled with a circuit. Treatment solutions have been diluted at rates from 10 to 260 ml / min. 

Figure 8. Example of toxicant preparation system.
Public Review Draft of December 17, 2014

Page 36 of 60

APPENDIX 2: Example Daily Calendar

Medaka Extend One-Generation Reproduction Test Daily Protocol

Enter Fertilization Date (F0 

Generation): 4/9/2012 Chemical:

Enter Start Date (move F0 

breeding pairs into system): 6/25/2012 Temp: 25o C

Event 

(1 row for each day of 

study) Day Date

Calendar 

Day Test Wk

Study 

Day

F1 

Wk

F1 

dpf

F2 

dpf

1st 

Tank 

to 

Feed

Brine 

Shrimp (dry 

wt.)/fish/day

Move breeding pairs into 

exposure system

Start exposure Mon 25-Jun-12 176 1 1 37 22.5 mg

Loading Phase Tue 26-Jun-12 177 2 5

Loading Phase Wed 27-Jun-12 178 3 17

Loading Phase Thu 28-Jun-12 179 4 13

Loading Phase Fri 29-Jun-12 180 5 7

Loading Phase Sat 30-Jun-12 181 6 42

Loading Phase Sun 1-Jul-12 182 7 16

Loading Phase Mon 2-Jul-12 183 2 8 39 22.5 mg

Loading Phase Tue 3-Jul-12 184 9 15

Loading Phase Wed 4-Jul-12 185 10 24

Loading Phase Thu 5-Jul-12 186 11 20

Loading Phase Fri 6-Jul-12 187 12 22

Loading Phase Sat 7-Jul-12 188 13 31

Loading Phase Sun 8-Jul-12 189 14 38

Loading Phase Mon 9-Jul-12 190 3 15 13 22.5 mg

Loading Phase Tue 10-Jul-12 191 16 38

Loading Phase Wed 11-Jul-12 192 17 15

Loading Phase Thu 12-Jul-12 193 18 9

Loading Phase Fri 13-Jul-12 194 19 32

Loading Phase Sat 14-Jul-12 195 20 29

siphon tanks and remove eggs Sun 15-Jul-12 196 21 8

Start F1 gen (Fill first set of 

incubators) Mon 16-Jul-12 197 4 22 1 0 1 22.5 mg

Second day of F1 egg 

collection if necessary ( Fill 

second set of incubators) Tue 17-Jul-12 198 23 1 13

Terminate F0 Generation Wed 18-Jul-12 199 24 2

Thu 19-Jul-12 200 25 3

Fri 20-Jul-12 201 26 4

Sat 21-Jul-12 202 27 5

Sun 22-Jul-12 203 28 6

Mon 23-Jul-12 204 5 29 2 7

Aeration turned off for first 

spawn incubators. Tue 24-Jul-12 205 30 8

Aeration turned off for second 

spawn incubators. Wed 25-Jul-12 206 31 9 35 0.5 mg

Thu 26-Jul-12 207 32 10 32 0.5 mg

Fri 27-Jul-12 208 33 11 27 0.6 mg
Public Review Draft of December 17, 2014

Page 37 of 60

Medaka Extend One-Generation Reproduction Test Daily Protocol

Enter Fertilization Date (F0 

Generation): 4/9/2012 Chemical:

Enter Start Date (move F0 

breeding pairs into system): 6/25/2012 Temp: 25o C

Event 

(1 row for each day of 

study) Day Date

Calendar 

Day Test Wk

Study 

Day

F1 

Wk

F1 

dpf

F2 

dpf

1st 

Tank 

to 

Feed

Brine 

Shrimp (dry 

wt.)/fish/day

Sat 28-Jul-12 209 34 12 26 0.7 mg

Sun 29-Jul-12 210 35 13 29 0.8 mg

Discard unhatched eggs from 

first spawn. Mon 30-Jul-12 211 6 36 3 14 16 1.0 mg

Discard unhatched eggs from 

second spawn. Tue 31-Jul-12 212 37 15 32 1.3 mg

Wed 1-Aug-12 213 38 16 36 1.7 mg

Thu 2-Aug-12 214 39 17 12 2.2 mg

Fri 3-Aug-12 215 40 18 40 2.8 mg

Sat 4-Aug-12 216 41 19 18 3.5 mg

Sun 5-Aug-12 217 42 20 6 4.2 mg

Mon 6-Aug-12 218 7 43 4 21 28 4.5 mg

Pool/Redistribute/Cull Tue 7-Aug-12 219 44 22 23 4.8 mg

Wed 8-Aug-12 220 45 23 26 5.2 mg

Thu 9-Aug-12 221 46 24 39 5.6 mg

Fri 10-Aug-12 222 47 25 11

Sat 11-Aug-12 223 48 26 19

Sun 12-Aug-12 224 49 27 12

Mon 13-Aug-12 225 8 50 5 28 36 7.7 mg

Tue 14-Aug-12 226 51 29 31

Wed 15-Aug-12 227 52 30 20

Thu 16-Aug-12 228 53 31 26

Fri 17-Aug-12 229 54 32 17

Sat 18-Aug-12 230 55 33 8

Sun 19-Aug-12 231 56 34 40

Mon 20-Aug-12 232 9 57 6 35 22 9.0 mg

Tue 21-Aug-12 233 58 36 7

Wed 22-Aug-12 234 59 37 18

Thu 23-Aug-12 235 60 38 19

Fri 24-Aug-12 236 61 39 7

Sat 25-Aug-12 237 62 40 42

Sun 26-Aug-12 238 63 41 36

Mon 27-Aug-12 239 10 64 7 42 11 11.0 mg

Tue 28-Aug-12 240 65 43 29

Wed 29-Aug-12 241 66 44 18

Thu 30-Aug-12 242 67 45 10

Fri 31-Aug-12 243 68 46 12

Sat 1-Sep-12 244 69 47 28

Sun 2-Sep-12 245 70 48 36

Mon 3-Sep-12 246 11 71 8 49 4 13.5 mg

Tue 4-Sep-12 247 72 50 22
Public Review Draft of December 17, 2014

Page 38 of 60

Medaka Extend One-Generation Reproduction Test Daily Protocol

Enter Fertilization Date (F0 

Generation): 4/9/2012 Chemical:

Enter Start Date (move F0 

breeding pairs into system): 6/25/2012 Temp: 25o C

Event 

(1 row for each day of 

study) Day Date

Calendar 

Day Test Wk

Study 

Day

F1 

Wk

F1 

dpf

F2 

dpf

1st 

Tank 

to 

Feed

Brine 

Shrimp (dry 

wt.)/fish/day

Wed 5-Sep-12 248 73 51 10

Thu 6-Sep-12 249 74 52 2

Fri 7-Sep-12 250 75 53 31

Sat 8-Sep-12 251 76 54 42

Sun 9-Sep-12 252 77 55 19

Fin Clip. Mon 10-Sep-12 253 12 78 9 56 35 22.5 mg

Tue 11-Sep-12 254 79 57 27

F1 Cull to pairs. Wed 12-Sep-12 255 80 58

F1 Cull to pairs. Thu 13-Sep-12 256 81 59

Fri 14-Sep-12 257 82 60

Sat 15-Sep-12 258 83 61

Sun 16-Sep-12 259 84 62

Mon 17-Sep-12 260 13 85 10 63 22.5 mg

Tue 18-Sep-12 261 86 64

Wed 19-Sep-12 262 87 65

Thu 20-Sep-12 263 88 66

Fri 21-Sep-12 264 89 67

Sat 22-Sep-12 265 90 68

Sun 23-Sep-12 266 91 69

Mon 24-Sep-12 267 14 92 11 70 22.5 mg

Tue 25-Sep-12 268 93 71

Wed 26-Sep-12 269 94 72

Thu 27-Sep-12 270 95 73

Fri 28-Sep-12 271 96 74

Sat 29-Sep-12 272 97 75

siphon tanks and remove eggs Sun 30-Sep-12 273 98 76

Start F1 fertility-fecundity 

assessment. Mon 1-Oct-12 274 15 99 12 77 22.5 mg

Tue 2-Oct-12 275 100 78

Wed 3-Oct-12 276 101 79

Thu 4-Oct-12 277 102 80

Fri 5-Oct-12 278 103 81

Sat 6-Oct-12 279 104 82

Sun 7-Oct-12 280 105 83

Mon 8-Oct-12 281 16 106 13 84 22.5 mg

Fertility-Fecundity 

Assessment Tue 9-Oct-12 282 107 85

Wed 10-Oct-12 283 108 86

Thu 11-Oct-12 284 109 87

Fri 12-Oct-12 285 110 88

Sat 13-Oct-12 286 111 89
Public Review Draft of December 17, 2014

Page 39 of 60

Medaka Extend One-Generation Reproduction Test Daily Protocol

Enter Fertilization Date (F0 

Generation): 4/9/2012 Chemical:

Enter Start Date (move F0 

breeding pairs into system): 6/25/2012 Temp: 25o C

Event 

(1 row for each day of 

study) Day Date

Calendar 

Day Test Wk

Study 

Day

F1 

Wk

F1 

dpf

F2 

dpf

1st 

Tank 

to 

Feed

Brine 

Shrimp (dry 

wt.)/fish/day

Sun 14-Oct-12 287 112 90

Mon 15-Oct-12 288 17 113 14 91 22.5 mg

Fertility-Fecundity 

Assessment Tue 16-Oct-12 289 114 92

Wed 17-Oct-12 290 115 93

Thu 18-Oct-12 291 116 94

Fri 19-Oct-12 292 117 95

Sat 20-Oct-12 293 118 96

Sun 21-Oct-12 294 119 97

Start F2 Generation, fill 

incubators Mon 22-Oct-12 295 18 120 15 98 0 22.5 mg

Terminate F1 Generation Tue 23-Oct-12 296 121 99 1

Wed 24-Oct-12 297 122 2

Thu 25-Oct-12 298 123 3

Fri 26-Oct-12 299 124 4

Sat 27-Oct-12 300 125 5

Sun 28-Oct-12 301 126 6

Mon 29-Oct-12 302 19 127 16 7

Aeration turned off for first 

spawn incubators. Tue 30-Oct-12 303 128 8

Aeration turned off for second 

spawn incubators. Wed 31-Oct-12 304 129 9

Thu 1-Nov-12 305 130 10

Fri 2-Nov-12 306 131 11

Sat 3-Nov-12 307 132 12

Sun 4-Nov-12 308 133 13

Discard unhatched eggs from 

first spawn. Mon 5-Nov-12 309 20 134 17 14

Discard unhatched eggs from 

second spawn. Tue 6-Nov-12 310 135 15
Page 40 of 60

APPENDIX 3: DNA Extraction from Fin Clips

3.1 Background.

In the MEOGRT, subadult medaka are assessed for the presence or absence of the DMY gene in order to 

1) setup up breeding pairs of one XX and one XY fish, and 2) provide DMY data for each of the fish 

sacrificed at this time. The first step in this process is to extract DNA from a tissue sample, typically a 

fin clip, of each fish. This can be done using commercially available kits that are based upon selective 

binding of DNA by a silica-gel-based membrane. Other methods are available, but use of a these kits 

provides consistently high quality DNA without the use of hazardous reagents.

3.2 Major Materials and Reagents.

DNA extraction kit (i.e. DNeasy Blood and Tissue Kit, Qiagen #69506)

Microcentrifuge and tubes

3.3 Protocol.

3.3.1 Tissue Collection. With the fish under anesthesia, remove a small piece (~1 mm square) 

of tail fin, place in a 1.5 ml microcentrifuge tube and immediately quench freeze in liquid 

nitrogen. Fins are stored at less than -20° C until further use. If tissue is going to be 

immediately processed, freezing can be omitted and the tissue can be processed as indicated 

below. 

3.3.2 DNA Extracton. Extract DNA from fin samples using the DNeasy Blood and Tissue Kit. 

Add 180 ul Buffer ATL to fin clip followed by 20 μl proteinase K, and vortex. Alternatively, 

Buffer ATL and proteinase K can be premixed immediately before sampling large numbers of 

fin clips and 200 μl added to fin clip. Incubate microcentrifuge tubes at 56°C until tissue is lysed 

(usually takes approximately 45 minutes but can be left overnight), vortex for 15 seconds, and 

add 200 μl Buffer AL before vortexing another time. Add 200 μl of 100% ethanol, and then 

vortex briefly. Pipet resulting mixture onto a DNeasy Mini spin column, centrifuge at greater 

than 6000 x g for 1 minute, and discard the flow-through/collection tube. Wash bound DNA 

with 500 μl Buffer AW1, centrifuge at greater than 6000 x g for 1 minute (discard flowthrough/collection tube) then wash with 500 μl Buffer AW2, centrifuge at greater than 20000 x g 

for 3 minutes to dry the column membrane (discard flow-through/collection tube). Place column 

in a new 1.5 ml microcentrifuge tube, pipet 100 μl Buffer AE onto the column, incubate at room 

temperature for 1 minute, and centrifuge at 6000 x g for 1 minute to elute DNA. DNA can be 

stored at less than -20°C until used.

 

Since the extracted DNA is only used to detect for the presence or absence of the DMY gene and 

no quantification is done, the concentration of the extracted DNA does not need to be assessed. 

In addition, there are sufficient quality assurance procedures in place during the actual DMY 

assessment that the quality of the extracted also does not need to be assessed. If experience with 

these types of DNA extraction kits is lacking, the concentration and quality of the DNA can be 

assessed by standard methods.
Page 41 of 60

APPENDIX 4: DMY Analysis of DNA 

4.1 Background.

In the MEOGRT, subadult medaka are assessed for the presence or absence of the DMY gene 

(GenBank: AB071534.1) in order to 1) setup up breeding pairs of one XX and one XY fish, and 

2) provide DMY data for each of the fish sacrificed at this time. After the DNA is extracted, the 

presence (or absence) of DMY, the medaka male determining gene, is assessed in each sample. 

DMY is present on the Y chromosome of every male somatic cell, but is not present in any of the 

female cells providing for a definitive test for genotypic sex: a DNA sample that is positive for 

the DMY gene is from a genotypic male while a DNA sample that is negative for the DMY gene 

is from a genotypic female (Matsuda et al. 2002). These results are independent of phenotypic 

sex which may have been altered by an EDC exposure. 

This protocol utilizes a Taqman® assay, a variant of real-time PCR to detect amplification of the 

DMY gene in each well of the 96-well plate. Starting at the designed primers, DNA polymerase 

moves downstream synthesizing a new strand from the template strand and via its 5’ exonuclease activity, the polymerase removes any bases that would impede its progress down the 

template strand. A Taqman® assay (5’ nuclease assay) uses this 5’ exo-nuclease activity in real 

time detection of PCR product. A Taqman® probe, present in the PCR mix, anneals to the 

template between the two primers in the path of the DNA polymerase as it progresses on the 

template strand. The Taqman® probe is a short sequence of DNA (~20 bp) with a reporter 

fluorophore on one end and a quencher on the other end. The close proximity of these two 

moieties allows interaction prohibiting the fluorescence of the reporter and thus its detection 

upon excitation within the real time instrument. However when the DNA polymerase cleaves 

the probe, both reporter and quencher are released into solution, and the distance between the 

two increases allowing the reporter to fluoresce upon excitation. Normally, the user is interested 

in the quantity of fluorescence from the reporter during the exponential phase of PCR as it is 

directly proportional to the amount of target sequence in the sample. However, in this 

application, NO QUANTIFICATION is attempted. The user is only interested in whether the 

DMY gene is present or absent. Any level of amplification above the defined threshold indicates 

the presence of the DMY gene, while no amplification indicates the absence of the DMY gene.

In addition, an internal control is included in the Master Mix that amplifies the 18S ribosomal 

subunit sequence (GenBank: X03205.1). Its presence is ubiquitous in eukaryotic cells, and thus 

provides a clear indication of successful PCR in each well. Or more to the point, if no 

amplification is present with this control, either there is no DNA present in the sample or there is 

inhibition of PCR by some contamination. 

There is another variation of the described protocol that instead of a primer/probe combination 

measuring fluorescence, only a primer set is used. This follows the typical protocol for 

traditional PCR followed by separation of the amplicon(s) with agarose gel electrophoresis. 

There are numerous references that can provide examples of protocols and possible primers, for 

example Padilla et al. 2009 and Shinomiya et al. 2004.
Public Review Draft of December 17, 2014

Page 42 of 60

4.2 Major Materials and Reagents.

 Universal PCR Master Mix (Applied Biosystems #4304437)

 18S Reagent (Applied Biosystems #4319413E)

 DMY Forward Primer (5’ TTC TGC CGC TGG AAA GAC 3’)

 DMY Reverse Primer (5’ TCT CTG GCG GAC CAT GAT 3’)

 DMY Probe (5’ FAM-CCA GTG CTT CAA ATG CGA GCA-BHQ 3’)

 Realtime thermal cycler (i.e. ABI 7500)

 96 well optical plates

 Centrifuge

4.3 Protocol.

4.3.1 Source DNA. DNA extracted using Appendix 3.

4.3.2 PCR. Again it should be stressed that while this protocol utilizes the ABI 7500 

thermocycler, which is an instrument used for QPCR, no quantification is done. In fact, 

this protocol is, in principle, a plus/minus assay. It maximizes throughput by using 96-

well plates and detecting amplification within each well, instead of running each product, 

post-PCR amplification, through gel electrophoresis. 

Prepare the following master mix keeping every reagent on ice shown in Table 1.

Table 1. Master Mix.

Reagents Volume per Sample

TaqMan® Universal PCR Master 

Mix 12.5 μl

18S reagent 0.25 μl

DMY Forward Primer (10 μM) 1.75 μl

DMY HTP Reverse (10 μM) 1.75 μl

DMY HTP Probe (1250 nM) 4.0 μl

Using a pre-chilled 96 well optical plate, load each well with 20.25 μl of Master Mix and 

add 4.75 μl of the DNA sample. Seal, vortex, and centrifuge plate at 2000 rpm for 5 

minutes to mix the samples with the Master Mix and remove any air bubbles. Air 

bubbles in the well will alter the fluorescent measurement. 
Public Review Draft of December 17, 2014

Page 43 of 60

Run the following thermal cycler program on the ABI 7500 as shown in Table 2.

Table 2. Thermal Cycler Program.

Step # Temperature Time

1 50 C 2 min

2 95 C 5 min

3 95 C 15 sec

4 60 C 1 min

5 Go to step 3 30 times

4.3.3 Interpretation of the Results. There are two detectors to consider when analyzing 

the results. The first shows the amplification associated with the 18S reagents (internal 

positive control, IPC). While the Ct value of the IPC will obviously be impacted by the 

concentration of the DNA, it may also be impacted by the amount of fluorescence in the 

other detector, the DMY detector, in particular if there is concurrent DMY amplification. 

In addition, the level of fluorescence (delta Rn) of the IPC detector may also be reduced if 

there is concurrent DMY amplification. This is because the primers and probe in the IPC 

are designed to have lower PCR efficiency than the DMY primers an probe, and therefore 

are out competed for “PCR resources”. 

A XX sample will have virtually no amplification in the DMY detector through the 30 

cycles of PCR, but there will be amplification in the IPC detector in a XX sample. If 

there is no amplification in either detector, there is a problem with the sample. The 

sample should be tested to verify the presence of high quality DNA, and reprocessed 

through the DNA extraction protocol if appropriate. A XY sample will have 

amplification in the DMY detector, and there will at least be some minimal evidence of 

amplification in the IPC detector if not normal amplification. Below is an example 

analysis of an XX fish (Figure 1) and an example analysis of an XY fish (Figure 2). 
Public Review Draft of December 17, 2014

Page 44 of 60

Figure 1. Example analysis of an XX fish. The blue plot is the amplification of the IPC detector, and 

the black plot is the DMY detector showing no amplification.

Figure 2. Example of the analysis of an XY fish (Figure 2). The blue plot is the amplification of the 

IPC detector which is somewhat reduced, and the black plot is the DMY detector showing clear 

amplification.
Public Review Draft of December 17, 2014

Page 45 of 60

4.4 References.

(1) Matsuda, M., Nagahama, Y., Shinomiya, A., Sato, T., Matsuda, C., Kobayashi, T., 

Morrey, C., Shibata, N., Asakawa, S., Shimizu, N., Hori, H., Hamaguchi, S., Sakaizumi, 

M., 2002. DMY is a y-specific dm-domain gene required for male development in the 

medaka fish. Nature 417: 559-563.

(2) Padilla, S., Cowden, J., Hinton, D.E., Yuen, B., Law, S., Kullman, S.W., Johnson, R., 

Hardman, R.C., Flynn, K., Au, D.W.T., 2009. Use of medaka in toxicity testing. Curr 

Protoc Toxicol. 39:1-36.

(3) Shinomiya, A., Otake, H., Togashi, K., Hamaguchi, S., Sakaizumi, M., 2004. Field 

survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild 

populations. Zool Sci 21:613-619.
Page 46 of 60

APPENDIX 5: Subadult Sampling – Grossing

5.1 Background.

In the MEOGRT after the DMY assessment and breeding pairs have been selected, the 

remaining subadult fish are sacrificed for growth, SSC, gonad phenotyping, and liver 

vitellogenin mRNA quantification. It is essential that the DMY status of these fish is 

maintained. If this information is lost during the selection of the breeding pairs, fresh tissue must 

be taken to repeat the DNA extraction and DMY analysis. 

5.2 Major Materials and Reagents.

 Dissecting microscope (with optional camera attached)

 Dissection tools including scissors

5.3 Protocol

5.3.1 Anesthesia. Place small numbers of fish in a solution of dilution water and 

buffered tricane methanesulfonate (MS-222) at 100 mg/L. 

5.3.2 Growth. Measure and record length to the nearest 0.1 mm and weight to the 

nearest mg. 

5.3.3 Grossing (Consult Figure 1). The first cut is a transverse cut done with high 

quality dissecting scissors between the operculum and the connection site of the pectoral 

fins. This cut is normally offset toward the pectoral fin. With experience, the prosector 

is able to make this cut so that the heart and the mesentery between it and the viscera 

stays with the head while the liver is exposed at the cranial opening of the carcass. The 

liver is separated from any connective tissue while it is teased away from the rest of the 

viscera through the opening made by the first cut (Figure 2).

A second cut is made caudal to the vent. The isolates the anal fin from the carcass. The 

number of anal fin papillae or SSC will be counted on the anal fin from each fish. At this 

time, an image of appropriate quality can be taken through a dissecting microscope of the 

anal fin or the anal fin can be preserved in an appropriate fixative like Davidson’s (note: 

do NOT use Bouin’s). If fixation is done, care must be taken so that the fin remains flat 

to allow for accurate counting. Regardless of the method, the number of anal fin papillae 

will be counted at a later date. 

A third cut is made on the tail fin to preserve some tissue for possible DMY analysis. 

The fin tissue should be immediately snap frozen in liquid nitrogen and kept frozen until 

processing.

The result of the grossing procedure is a dissected liver for vitellogenin mRNA analysis, 

a head that can be discarded, a tail with the anal fin for counting of the anal fin papillae, 

and a portion of the tail fin for possible DMY analysis (if needed at a later date).
Public Review Draft of December 17, 2014

Page 47 of 60

Figure 1. Cuts made during grossing of subadult samples.

Figure 2. Dissection of the liver at the subadult sampling.
Page 48 of 60

APPENDIX 6: Counting Anal Fin Papillae

6.1 Background. 

This is a method to count the number of anal fin papillae. Under normal circumstances, 

only sexually mature male medaka have papillae, which develop on the joint plates of certain 

anal fin rays as a secondary sexual characteristic, providing a potential biomarker for EDC 

exposure.

6.2 Major Materials and Reagents. 

 Dissecting microscope (with optional camera attached)

 Fixative (if not counting from image)

6.3 Protocol.

After the grossing procedure (previously documented), the anal fin should be imaged to 

allow for convenient counting of anal fin papillae. While imaging is the recommended method, 

the anal fin can be fixed with Davidson’s fixative or other appropriate fixative (Bouin’s is not 

recommended) for approximately 1 minute. It is important to keep the anal fin flat during 

fixation to allow for easier counting of papillae. The carcass with the anal fin can be stored in 

Davidson’s fixative or other appropriate fixative until analyzed. Count the number of joint plates 

(see Figure 1) with papillae which protrude from the posterior margin of the joint plate.

Figure 1. Anal fin papillae.
Page 49 of 60

APPENDIX 7: RNA Extraction from Medaka Liver

7.1 Background.

In the MEOGRT, subadult medaka are sacrificed for various endpoints. One of these 

endpoints is the number of copies of the vitellogenin 1 gene (Vtg1) per ng of total RNA in the 

liver determined by quantitative PCR (QPCR). QPCR is highly sensitive and has a large 

dynamic range (~7 orders of magnitude), making it an ideal tool for efficiently assessing 

concentrations of a target gene, in this case VTG1. In order to do this analysis, RNA must be 

extracted from the liver of subadult medaka. The following is intended to provide guidance for 

the proper extraction and handling of hepatic RNA in support of the MEOGRT.

7.2 Major Materials and Reagents.

 RNeasy Protect Mini Kit (Qiagen #74126)

 Microcentrifuge

 Microcentrifuge tubes (2.0 or 1.5 ml)

 NanoDrop ND-1000 (Thermo Scientific)

7.3 Protocol.

7.3.1 Source Tissue. As per the dissection protocol of subadult medaka within the 

MEOGRT, the liver that is the source tissue for the RNA extraction protocol has been 

preserved in RNAlater (Qiagen #76104). 

7.3.2 RNA Extraction with RNeasy Protect Mini Kit (Qiagen #74126). The 

manufacturer’s instructions are followed (RNeasy Mini Handbook 4th edition, September 

2010) for animal tissues with the following exception. Disruption of a RNAlater

preserved liver is done in a 2 ml microcentrifuge tube with the liver, 600 μl of Buffer 

RLT, and a moderate number (~20 to 100) of 0.5 mm glass beads (BioSpec Products 

#11079105). Disruption is completed by vortexing for 5 minutes on a Disruptor Genie 

(Scientific Industries). This homogenate is centrifuged for 3 minutes at full speed and the 

supernatant is pipetted into a new microcentrifuge tube. At this point, the manufacturer’s 

instructions are followed exactly.

7.3.3 Assessment of the Quality and Concentration of the Extracted RNA. For proper 

analysis of the number of copies of the Vtg1 gene, the concentration of the extracted 

RNA must be determined. In addition, it is highly recommended that the quality of the 

RNA is determined. Both of these can be accomplished by analyzing a 2 μl aliquot of 

each RNA sample on a spectrophotometer like the NanoDrop ND-1000 (Thermo 

Scientific). The NanoDrop will provide a concentration in ng of RNA per μl of sample 

and ratios of the absorbance at 260/280 nm and 260/230 nm. The 260/280ratio should be 

~2.0 and the 260/230 ratio should be 2.0 – 2.2. If either ratio is appreciably lower it may 

indicate the presence of contaminants. It is recommended that offending samples are rerun through the RNeasy Mini Kit, a similar product, or another purification protocol to 

further clean-up the RNA to remove this contamination.
Page 50 of 60

APPENDIX 8: Preparation of RNA for Standard Curve Used during VTG1 QPCR

8.1 Background.

In the MEOGRT, subadult medaka are sacrificed for various endpoints. One of these 

endpoints is the number of copies of the vitellogenin 1 gene (Vtg1; GenBank: AB064320.1) per 

ng of total RNA in the liver determined by quantitative PCR (QPCR). QPCR is highly sensitive 

and has a large dynamic range (~7 orders of magnitude), making it an ideal tool for efficiently 

assessing concentrations of a target gene, in this case VTG1. 

For absolute quantification, RNA that contains the Vtg1 sequence targeted by the primers and is 

only minimally larger than this amplicon must by synthesized. Ideally this RNA would have the 

identical sequence and number of base pairs as the amplicon produced by the primers used for 

Vtg1 QPCR. The intent is for this “standard RNA” to be replicated with the same efficiency as 

the target sequence within the Vtg1 mRNA extracted from livers during the MEOGRT protocol. 

This ideal RNA would be amplified during QPCR at an identical rate as the target sequence in 

actual samples. In practice, primers were designed just outside the Vtg1 amplicon that would 

produce cDNA that is complimentary to the Vtg1 target sequence. In addition, the forward 

primer contained the T7 promoter sequence which allows for use of RNA polymerase that 

recognizes the T7 promoter sequence to make RNA copies of this cDNA template. 

The concentration of this RNA and its size in base pairs are used to calculate the number of 

copies of the Vtg1 target sequence present per unit volume. The RNA is then serially diluted to 

known copy number per unit volume (μl) in order to produce a standard curve which is used to 

determine the absolute copy number of VTG1 in the RNA from each liver sample obtained in the 

MEOGRT.

In the following protocol, the use of specific reagents and equipment are detailed. While these 

have been verified to work appropriately, reagents and equipment from other manufacturers can 

be substituted as long as their appropriateness is verified. 

8.2 Major Materials and Reagents.

 RNase/DNase free water (Qiagen #129114)

 NanoDrop ND-1000 (Thermo Scientific)

 Superscript II (Invitrogen #18064-022)

 AmpliTaq Gold (Applied Biosystems #4311806)

 MEGAScript (Ambion AM1333)

 Bioanalyzer 2100 (Agilent Technologies)

8.3 Protocol.

8.3.1 Source RNA. Extract RNA from liver of adult female medaka that are known 

successful spawners. Follow MEOGRT Protocol: RNA Extraction of Liver from 

Medaka. Ensure that the RNA is of good quality as described in the protocol and has a 

concentration greater than 200 ng/μl.
Public Review Draft of December 17, 2014

Page 51 of 60

8.3.2 Synthesizing cDNA from liver RNA. Adjust the RNA concentration to 

200 ng/μl with RNase/DNase free water. It is recommended this water is purchased from 

a qualified supplier (example: Qiagen #129114). It is recommended that several samples 

of RNA are used in case problems arise during the remainder of this protocol.

Prepare the following reaction mix (Table 1):

Table 1. Reaction Mix.

Reagent Per Sample 

Volume

RNase/DNase free water 3 μl

RNasing (Promega N2511) 1 μl

Oligo d(T) (Promega C110A or C1101) 1 μl

5x RM buffer (in Superscript II Kit, Invitrogen) 4 μl

0.1M DDT (in Superscript II kit, Invitrogen) 2 μl

dNTP (10mM, Promega U1511) 4 μl

Reverse transcriptase (Superscript II, Invitrogen 

#18064-022)

1 μl

Proceed with reverse transcription for 1 hour at 37°C. Each sample consists of 16 μl of 

the above reaction mix + 4 μl RNA at 200 ng/μl from above. This procedure will 

produce cDNA from the mRNA present in the RNA samples. Since Vtg1 mRNA is 

highly represented in the RNA samples, the cDNA produced from each of those samples 

will be well populated with Vtg1 cDNA. This procedure is the reverse transcription step 

of RT-PCR. 

8.3.3 Amplifying the Vtg1 cDNA. The cDNA produced above is a cDNA representation 

of the various mRNA transcripts that were present in the original female liver RNA 

samples. The next step is to selectively amplify the VTG1 cDNA, that is, to perform the 

amplification step of RT-PCR with primers specific for VTG1. In addition, the T7 

promoter sequence (TAA TAC GAC TCA CTA TAG GGA GA) for T7 RNA 

polymerase is also included on the 5’ end of the forward primer so that in future steps the 

amplified VTG1 cDNA can be transcribed into RNA. 

The amplification step is done with the following reaction mix (Table 2) and thermal 

cycler profile (Table 3).
Public Review Draft of December 17, 2014

Page 52 of 60

Table 2. Amplification Reaction Mix.

Note: Underlined is the T7 promoter sequence.

Table 3. Thermal Cycler Profile.

Step Temperature Time

1 95°C 10 minutes

2 94°C 30 seconds

3 58°C 1 minute

4 72°C 1 minute

5 Go to 2, 34 cycles

8.3.4 Convert Vtg1 cDNA to RNA. The amplified product from the RT-PCR needs to

be used to synthesize RNA via RNA polymerase. Specifically, T7 RNA polymerase is 

used which recognizes the T7 promoter sequence that has been incorporated into the 

Vtg1 amplicon with its inclusion at the 5’ end of the forward primer. In this way, RNA 

polymerase will synthesize RNA that is similar to the target sequence in VTG1 mRNA, 

and therefore the amplification efficiency in the quantitative PCR of sample RNA will be 

the same as the RNA for the standard curve. The difference between the standard RNA 

and the target sequence within liver RNA samples is the number of base pairs. The 

standard RNA primers are just outside the primers for quantification.

To convert the cDNA to RNA, first prepare the following reaction mix (Table 4).

Reagent Per Sample Volume

RNase/DNase free water 25.36 μl

10x RM buffer (from AmpliTaq Gold, Applied Biosystems) 4 μl

MgCl2 (from AmpliTaq Gold) 2.24 μl

Vtg1 Standard Forward 

(TAATACGACTCACTATAGGGAGATTCCTCGGATACGGCACAAT) 2 μl

Vtg1 Standard Reverse 

(TAGACAGCTTTGCTGTAACGTAAGC) 2 μl

AmpliTaq Gold (Applied Biosystems, #4311806) 0.4 μl
Public Review Draft of December 17, 2014

Page 53 of 60

Table 4. Reaction Mix to Convert cDNA to RNA.

Incubate the above for 1 hour at 37°C. Add 1 μl of DNase (Invitrogen #18068-015) and 

incubate for 15 minutes at 37°C. Clean the RNA from free nucleotides, buffer 

components, and enzymes with a MEGAclear Kit (Ambion #AM1908) following the 

manufacturer’s instructions.

8.3.5 Determine the quantity and quality of the RNA. Quantify the RNA on a 

NanoDrop ND-1000 (Thermo Scientific) following the manufacturer’s instructions. This 

will provide a concentration in μg/μl. Calculate the number of copies per μl using the 

following equations from Roche Molecular Biochemicals (Technical Note #LC 11/2000)

(Table 5). 

Table 5. Calculation of Number of dsDNA, ssDNA, or ssRNA Copies

For average molecular weight of: Use this calculation

dsDNA (number of base pairs) X (660 daltons/base pair)

ssDNA (number of base pairs) X (330 daltons/base pair)

ssRNA (number of base pairs) X (640 daltons/base pair)

Example Calculation (For Demonstration Purposes Only):

General Formula:

MW = molecular weight [g/mol]

1 mol = 6 X 1023 molecules (= copies)

Reagent Per Sample Volume

cDNA (from above) 12 μl

10x buffer (from MEGAScript) 2 μl

10mM ATP (from MEGAScript) 1 μl

10mM CTP (from MEGAScript) 1 μl

10mM GTP (from MEGAScript) 1 μl

10mM UTP (from MEGAScript) 1 μl

RNA polymerase (Ambion MEGAScript, AM1333) 2 μl
Public Review Draft of December 17, 2014

Page 54 of 60

Example: For CycA plasmid that has a concentration of 152 ng/μl = 1.52 X 10–7g/μl 

and a total size of 3,397 bp:

MW = 3,397 bp X 660 daltons/bp = 2.24 X 106daltons

1 mol = 2.24 X 106g

1 mol = 6 X 1023 molecules (= copies)

The quality of the RNA should be determined via a Bioanalyzer 2100 (Agilent 

Technologies) using a RNA 6000 Nano Kit (Agilent Technologies #5067-1511) 

following the manufacturer’s instructions. The Vtg1 standard RNA should be 612 base 

pairs. As a rule of thumb, the RNA should also have a RNA Integrity Number 

(calculated by the Bioanalyzer software) of ~7 or greater. 

Dilute the RNA based upon its concentration in copies per μl to produce a series that is 

diluted a factor of 10 between each step, for instance 1010, 109, 108, etc. To do this, since 

the copy numbers for the standards are based upon loading 4 μl of standard RNA into 

each well that will be used to generate the standard curve, the STD10 (1 X 1010) would 

have a concentration of 2.5 X 109copies/ μl. For instance, if the standard RNA has a 

concentration of 2.3 X 1011 copies/μl then this would be diluted to 2.5 X 109copies/μl by 

adding 91 μl of RNase/DNase free water to every 1 μl of the standard RNA. This 2.5 X 

109copies/μl standard is called STD10 since when used as prescribed with 4 μl per well, 

there is 1.0 X 1010 copies per well. The other standards (STD9 through STD3) are 

serially diluted from STD10 with the following formula maintained: STDN = 2.5 X 10N-1

copies/μl.
Page 55 of 60

APPENDIX 9: QPCR to Quantify Vtg1 mRNA in Liver

9.1 Background. 

In the MEOGRT, subadult medaka are sacrificed for various endpoints. One of these 

endpoints is the number of copies of the vitellogenin 1 gene (Vtg1; GenBank: AB064320.1) per 

ng of total RNA in the liver determined by quantitative PCR (QPCR). QPCR is highly sensitive 

and has a large dynamic range (~7 orders of magnitude), making it an ideal tool for efficiently 

assessing concentrations of a target gene, in this case VTG1. RNA was extracted from the liver 

of subadult medaka and RNA was produced for standards that generate a standard curve for 

absolute quantification of Vtg1 mRNA using Taqman-based QPCR.

The following is a brief summary of the Taqman protocol. Starting at the designed primers, 

DNA polymerase moves downstream synthesizing a new strand from the template strand and via 

its 5’ exo-nuclease activity, the polymerase removes any bases that would impede its progress 

down the template strand. A Taqman® assay (5’ nuclease assay) uses this 5’ exo-nuclease 

activity in real time detection of PCR product. A Taqman® probe, present in the PCR mix, 

anneals to the template between the two primers in the path of the DNA polymerase as it 

progresses on the template strand. The Taqman® probe is a short sequence of DNA (~20 bp) 

with a reporter fluorophore on one end and a quencher on the other end. The close proximity of 

these two moieties allows interaction prohibiting the fluorescence of the reporter and thus its 

detection upon excitation within the real time instrument. However when the DNA polymerase 

cleaves the probe, both reporter and quencher are released into solution, and the distance 

between the two increases allowing the reporter to fluoresce upon excitation. The quantity of 

fluorescence from the reporter during the exponential phase of PCR is directly proportional to 

the amount of target sequence in the sample. During PCR, amplicon production goes through 

three phases: (1) the exponential phase where doubling of amplicon is occurring every cycle and 

the reaction is very specific and precise; (2) the linear phase where reagents are becoming rate 

limiting, the reaction slows and amplicons may start to degrade; and (3) plateau phase where the 

reaction has stopped with no additional product formed (Figure 1). Again, during real-time PCR, 

fluorescent measurements are taken during the exponential phase while with traditional PCR, the 

reaction is often terminated in the plateau phase and amplicons detected with a gel.

9.2 Major Materials and Reagents.

 “Unknown” RNA (extracted from liver)

 “Standard” RNA (produced in MRT Protocol: Preparation of RNA for Standard Curve 

Used during Vtg WPCR)

 Taqman EZ RT-PCR Reagents (Applied Biosystems #N8080236)

 ABI 7500 (Applied Biosystems)

9.3 Protocol

9.3.1 Source RNA. There are two sources of RNA to be used in this protocol. First, 

follow the “MRT Protocol: RNA Extraction of Liver from Medaka” (Appendix 7) to 

extract RNA from subadult medaka. Secondly, follow “MRT Protocol: Preparation of 
Public Review Draft of December 17, 2014

Page 56 of 60

RNA for Standard Curve Used during Vtg QPCR” (Appendix 8) to produce standard 

RNA. In both cases, ensure that the RNA is of good quality as described in the specific 

protocol and of known concentration either ng/μl or copies/μl.

9.3.2 QPCR. Prepare the following master mix keeping every reagent on ice as shown in 

Table 1.

Table 1. QPCR Master Mix.

Taqman EZ RT-PCR Reagents

(Applied Biosystems # N8080236) μl/reaction

5x Taqman Buffer 6.0

Mn2+ acetate 3.6

dATP 0.9

dGTP 0.9

dCTP 0.9

dUTP 0.9

Vtg1 Forward (10 μM)

5’-AGGCAGTTTCTAAGGGCGAAC-3’

1.5

Vtg1 Reverse (10 μM)

5’-TGAATGGGCATAATCTTTGTGATT-3’

1.5

Vtg1 Probe (1250 nM)

5’-fam-TTTGGGAAATGCAAGACACCCTA-bhq-3’

4.8

rtTH polymerase 1.2

AmpErase 0.2

RNase/DNase-free water 3.6

Total 26

Add 26 μl of the master mix to each well and then 4 μl of the RNA sample from subadult 

liver or standard RNA to each well. Unknown samples should be run minimally in 

duplicate while standard RNA should be run in triplicate. In addition to samples and 

standards, appropriate technical controls should be included. These should minimally 

include “no template controls” and a liver mRNA sample that has previously been 

quantified. Other technical controls can be included as warranted. The 96-well optical 

plate is sealed, vortexed, and centrifuged at ~2000 rpm for ~ 5 minutes to remove air 

bubbles.

QPCR is performed on an ABI 7500 (Applied Biosystems) with the following thermal 

profile (Table 2).

Table 2. QPCR Thermal Profile.

Temperature Time Cycles Temperature Time Cycles

50°C 2 minutes

1

95°C 15 seconds 45 60°C 30 minutes 60°C 1 minute

95°C 5 minutes
Public Review Draft of December 17, 2014

Page 57 of 60

An example plate layout is provided in Table 3.

Table 3. Example Plate Layout.

NTC = no template control; known = liver RNA sample with known copies/μl; STD3-10 = 

standard RNA

9.3.3 Interpretation of QPCR. The guidance provided here is not meant to supersede or 

replace the manufacturer’s instructions on the analysis of QPCR data on the 7500 system. 

The ABI 7500 System Software is usually able to automatically set both the baseline 

range and the critical threshold (Ct) value. If the software is unable to do this 

automatically, a knowledgeable scientist will need to input the appropriate values for 

these parameters into the software. Once these parameters are set, the ABI 7500 System 

Software will generate a standard curve based upon the inputed copy number for each 

standard RNA and the corresponding Ct value. The software will also calculate the copy 

number, the mean copy number of the replicated sample and the standard deviation of the 

replicated sample for each unknown sample based upon its specific Ct value. It is 

recommended that the CV% of each replicated sample is less than 10%. In addition, 

there should be no amplification in the “no template control” samples and the known 

sample should fall within +/- 10% of its running mean from previous analyses. 

9.3.4 Data Reporting. All raw data should be maintained. Assuming the standard 

deviation of any specific sample was acceptable, the mean copy number of Vtg1 mRNA 

per μl is recorded for each specific sample. For each sample, there are two pieces of data 

that are used: 1) the copy number per μl just derived and 2) the concentration in ng/μl of 

the liver RNA (obtained in MRT Protocol: RNA Extraction of Liver from Medaka). This 

data is used to calculate the Vtg1 copy number per ng of total RNA which is the 

information to be reported from this protocol. In addition, performers of this analysis 

should be familiar with the published MIQE guidelines (Bustin et al. 2009; Shipley 2011) 

and all pertinent information required by the MIQE guidelines should be recorded and 

provided in MEOGRT reports. 

1 2 3 4 5 6 7 8 9 10 11 12

A NTC STD3 STD3 STD3 Sample 

3

Sample 

3

Sample 11 Sample 

11

Sample 

19

Sample 

19

Sample 

27

Sample 

27

B NTC STD4 STD4 STD4 Sample 

4

Sample 

4

Sample 12 Sample 

12

Sample 

20

Sample 

20

Sample 

28

Sample 

28

C known STD5 STD5 STD5 Sample 

5

Sample 

5

Sample 13 Sample 

13

Sample 

21

Sample 

21

Sample 

29

Sample 

29

D known STD6 STD6 STD6 Sample 

6

Sample 

6

Sample 14 Sample 

14

Sample 

22

Sample 

22

Sample 

30

Sample 

30

E

Sample

1

STD7 STD7 STD7 Sample 

7

Sample 

7

Sample 15 Sample 

15

Sample 

23

Sample 

23

Sample 

31

Sample 

31

F

Sample 

1

STD8 STD8 STD8 Sample 

8

Sample 

8

Sample 16 Sample 

16

Sample 

24

Sample 

24

Sample 

32

Sample 

32

G

Sample 

2

STD9 STD9 STD9 Sample 

9

Sample 

9

Sample 17 Sample 

17

Sample 

25

Sample 

25

Sample 

33

Sample 

33

H

Sample 

2

STD10 STD10 STD10 Sample 

10

Sample 

10

Sample 18 Sample 

18

Sample 

26

Sample 

26

Sample 

34

Sample 

34
Public Review Draft of December 17, 2014

Page 58 of 60

9.4 References.

(1) Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, 

R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., and Wittwer, C.T., 2009. 

The MIQE guidelines: minimum information for publication of quantitative real-time 

PCR experiments. Clin Chem 55: 611-622. 

(2) Shipley, G., 2011. The MIQE Guidelines Uncloaked. Polymerase Chain Reaction 

Troubleshooting and Optimization: The Essential Guide, 151.
Page 59 of 60

APPENDIX 10: Data Reporting Templates

10.1 Background. 

To expedite data reporting and statistical analysis, especially if Agency provided 

statistical tools are used, it is recommended that common templates for data reporting be used. 

These templates take the form of Excel spreadsheets that can easily be converted into the CSV 

file format which is required by the Agency provided statistical tool. Example formats are 

provided below and electronic versions of all templates can be accessed at the EPA EDSP 

website.

10.2 Data Templates

10.2.1 Template for Recording Fecundity and Fertility. Table 1 shows a portion of 

the electronic data sheet for recording both fecundity and fertility that represents the 

complete dataset for one replicate. All fields except “Study” are required to properly 

analyze the data. Other fields containing metadata can be added as needed. An 

electronic version at the EPA EDSP website. To be entered into StatCHARRMS, the 

data must be in the CSV format which can easily be converted within Excel.

Table 1. Example for Recording Fecundity and Fertility Data

Study Date Treatment Replicate Fecundity Fertile Eggs

PTOP-03 10/1/2012 1 A 7 7

PTOP-03 10/2/2012 1 A 7 7

PTOP-03 10/3/2012 1 A 8 8

PTOP-03 10/4/2012 1 A 11 11

PTOP-03 10/5/2012 1 A 13 13

PTOP-03 10/6/2012 1 A 15 15

PTOP-03 10/7/2012 1 A 8 8

PTOP-03 10/8/2012 1 A 16 14

PTOP-03 10/9/2012 1 A 21 21

PTOP-03 10/10/2012 1 A 15 15

PTOP-03 10/11/2012 1 A 16 16

PTOP-03 10/12/2012 1 A 13 13

PTOP-03 10/13/2012 1 A 12 12

PTOP-03 10/14/2012 1 A 12 9

PTOP-03 10/15/2012 1 A 11 10

PTOP-03 10/16/2012 1 A 24 23

PTOP-03 10/17/2012 1 A 20 18

PTOP-03 10/18/2012 1 A 20 20

PTOP-03 10/19/2012 1 A 16 16

PTOP-03 10/20/2012 1 A 17 16

PTOP-03 10/21/2012 1 A 8 5
Public Review Draft of December 17, 2014

Page 60 of 60

10.2.2 Template for Recording Endpoint Information. Table 2 illustrates a portion of 

the electronic data sheet for recording endpoint information. Additional fields for 

metadata can be added as necessary. An electronic version is available at the EPA EDSP 

website.

 

Table 2. Template for Recording Endpoint Information.

Study ID Treatment Generation Genotypic_Sex Vtg SSC Age Weight Length

PTOP T1A1 1 F1 Male 1.50E+03 86 8_Week 183 22

PTOP T1A3 1 F1 Female 6.60E+06 0 8_Week 182 23

PTOP T1A4 1 F1 Female 1.20E+06 0 8_Week 196 24

PTOP T1A5 1 F1 Male 2.70E+03 61 8_Week 199 21

PTOP T1A6 1 F1 Male 8.50E+02 59 8_Week 192 23

PTOP T1A7 1 F1 Male 1.10E+03 80 8_Week 181 22

PTOP T1A8 1 F1 Male 1.20E+03 38 8_Week 115 25

 

10.2.3 Template for Recording Pathology Information. Table 3 illustrates a portion of 

the electronic data sheet for recording pathology information. Only two fields of 

pathology diagnoses are shown here, however the pathology guidance contains many 

more potential pathologies. Additional fields for metadata can also be added as 

necessary. An electronic version is available at the EPA EDSP website. 

Table 3. Template for Recording Pathology Information.

ID Gen Treatment Rep DMY Age Gon_Phenotype Gon_Stage

T1A1 F0 1 A Male 16_wk 1 3

T1A2 F0 1 A Female 16_wk 5 3

T1B1 F0 1 B Male 16_wk 1 3

T1B2 F0 1 B Female 16_wk 5 3

T1C1 F0 1 C Male 16_wk 1 2

T1C2 F0 1 C Female 16_wk 5 2

T1D1 F0 1 D Male 16_wk 1 2

T1D2 F0 1 D Female 16_wk 5 3

T1E1 F0 1 E Male 16_wk 1 1

T1E2 F0 1 E Female 16_wk 5 3

T1F1 F0 1 F Male 16_wk 1 2

T1F2 F0 1 F Female 16_wk 5 3