### 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 sub mission 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 specificall y 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 t hat 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 guidanc e, 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 t hat 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 acces s 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: T his 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) aff ected 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 an d 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

aff ects 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 eff ects.

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 perf ormed. 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 detai led 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,

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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 s tage 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

rela tionship 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 OCSP P 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 g eneration.

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., hypothalamus

pituitary

- 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 s uch 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 Guidelin e 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), histopath ological ( 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 addi tional 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

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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 f

ourth 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

co mplete 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 tr eatment 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 s olvent 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 devel opment 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 opt imally 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 fe cundity (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

collec ted in the same manner with the omission of hatching success.

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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 sta tus 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 considerat ion 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 F 1 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 F 0 F 1

 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 F 0 F 1

Hatch F 0 F 1

Survival F 1 F 1 F 1

Growth F 0 F 1 F 1

Vitellogenin F 1

Secondary sex F 1 F 1

Histopathology F 1

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

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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 F 0 F 1 F 2

Fertility F 0 F 1 F 2

Hatch F0 F 1

Survival F1 F 1 F 1 F 2 F 2 F 2

Growth F 0 F 1 F 1 F 2 F 2

Vitellogenin F 1 F 2

Secondary sex F 1 F 1 F 2 F 2

Histopathology F 1 F 2

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; Yok ota 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 te st 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 t o 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

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Page 8 of 60 and illumination similar to those used in the test. In addition, each breeding pair of F 0

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 p airs 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 inc lusion 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 init iated 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 pa irs 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 rep roduction 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, SO 4 

### ), pesticides, total organic carbon and suspended solids

sho uld 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.

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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 system s 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 cell s, 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 so lvent 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 aquari a 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 spec ified 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 d ry 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

temperat ure 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 replic ate 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 sys tem.

(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 aqu aria 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 th e test substance may be difficult to test in aquatic systems include: high log octanol

- water partitioning coefficients (log K
ow 

### ), 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

- by

case 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

succes s 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

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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 a void 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 min imally 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 li fe 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 lowe r 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 Su bstance. 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 subs tance 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 spectrosc opy (GC

- MS), HPLC (high performance liquid chromatography) with an appropriate detector, or ion

- chromatography with conductivity

detection.

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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 F 0 ~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 con trol 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)



### * I f 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 m et 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 t o 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 e ggs from the female and by siphoning all the eggs from the bottom of each aquarium. The eggs from each

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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 hat ch. 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 co ntrol 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

treatme nt 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) nee d 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 f or 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 8 th 

### day post

fertilization) , 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 hatchli ngs 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 t wice 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 16 th 

### day post

fertilization).

(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

indiv idual’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 on e 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 con trol(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 t o 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 th e 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 (Figu re 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 incl uding: 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 gross ing 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 f rom 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 t he 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 prev iously 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 n eeded) 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 16 th


### 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 gener ation.

(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, kidn eys 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 insta nce, 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 guida nce.

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

bre eding 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 follow ed 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 w eeks 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 addit ional 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 genera tion 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, c ounted, 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 detai led 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 o n 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 s ystematically 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 in festations, 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 m ore 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 replicat es 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

h atching success of each replicate incubator. As detailed previously, embryos that have not hatched by twice the median control day of hatch (typically the 16 th


### 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 s amples 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) ar e 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 X Y) 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 We eks 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 ass essed 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 co ntrol 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 e gg 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 8 th 

### day post

- fertilization), approximat ely 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 16 th 

### day post

- fertilization). The total number of hatchlings in each replicate is recorded to determine hatch success.

Optional F2 Gene ration. 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). Si nce 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 ide ntical to the F1 generation.

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 gro wth 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 accoun t 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 t reatment 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 ot her 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 concen tration 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 t o 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 dilu ted 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 substanc e 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 clea ning 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 an d 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

res iduals 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 measu red 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 continuousl y 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 delivere d 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 the m 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. T he 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 fro m 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 (handli ng) 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 ac hieve 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 agai nst 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 i n 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 m ay 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 Fi gure 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 wou ld make a one

tailed 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 ( R ao

- S cott C ochran

A rmitage b y S lices) 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 conc entrations, 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 determ ining 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 transfor med 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 q uadratic 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 th e 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 tr ansforms 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 arcsine

square root transformation is recommended for the data on percen t 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 d one 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 treatme nts 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 te st 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 b e 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

an d 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 cens ored 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 ch oice 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 tes t, 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 a nd 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 inf

ormation, 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, alkali nity, 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 dilutio n 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 u sed; 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 tab ular 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 whi ch 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 identi fication of dose

response 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 pro vide 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 concentr ation 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 mort ality 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

. , Hamm ermeister 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

- 56 3.

(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

. , T adokoro 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

. , Kobayash i 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 ( O ryzias 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 Calenda r

............................................. ............. 36

(3) DNA Extraction from Fin Clips

....................................... ......... 40

(4) DMY Analysis of DNA

................................................... ........ 41

(5) Subadult Sampling

– Grossing

.......................................... ......... 4 6

(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) Da ta 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 an d 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 importa nt 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 t o 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 incubat or.

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 whi le 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 botto m, 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 v alves 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 F 1 

### 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 chambe rs 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 t he 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 tem perature 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 d eliver flows up to 50 ml/min/channel. Delivery lines should be sterilized on a set maintenance schedule to control in

line 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 treatmen t flow is controlled by the

spe d 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 experimenta l 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 ta nks.

 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 re servoir. 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 s wapping 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 t o 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: 25 o 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: 25 o 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: 25 o 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: 25 o 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 th at 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 extractio n 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 immed iat ely quench freeze in liquid nitrogen. Fins are stored at less than

- 20° C until fu rther 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 f rom 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% etha nol, 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 flow

through/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 tu be, 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 prese nce 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 Backgro und

.

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. Aft er 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 definitiv e 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’ exo

nuclease activity, the polymerase removes any bases that would impede its progress down the template strand. A Taqman® a ssay (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 d etection 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 excitati on. 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 a ttempted. 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

mea suring 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 pos sible 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 m ix 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 C t 

### value of the IPC will obviously be impacted by the

concentration o f 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 R
n 

### ) 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 no rmal 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 s omewhat 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., Togas hi, K., Hamaguchi, S., Sakaizumi, M., 2004. Field survey of sex

- reversals in the medaka, Oryzias latipes : genotypic sexing of w ild 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

m aintained. 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)

 Disse ction 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 w eight 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 pectora l 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 m icroscope 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 metho d, 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 fix ative (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 fix ative 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 ( Vtg 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. 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 RNA later (Qiagen #76104).

7.3 .2 RNA Extraction with RNeasy Protect Mini Kit (Qiagen #74126)

. The manufacturer’s instructions are followed (RNeasy Mini Handbook 4 th 

### edition, September 2010) for animal tissues with the following exception. Disruption of a RNA later preserved liver is do ne 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 E xtracted 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 a nalyzing 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 260/28 0ratio 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 re

run through the RNeasy Mini Kit, a similar product, or ano ther 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 endpoint s. 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 magnitu de), 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 s ynthesized. 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 wit hin 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 wor k 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 me daka that are known

successful spawners. Follow MEOGRT Protocol: RNA Extraction of Liver from

Medaka. Ensure that the RNA is of good quality as descr ibed 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 concentrat ion 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 proto col.

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 repr esented 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 repr esentatio n 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 foll owing r eaction 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 whic h recognizes the T7 promoter sequence that has been incor porated into the Vtg1 amplicon with its inclusion at the 5’ end of the for ward primer. In this way, RNA polymerase will synthesize RNA that i s similar to the target sequence in VTG1 mRNA, and therefore the amplification efficie ncy in the quantitative PCR of sample RNA will be

the same as the RNA for the s tandard curve. The difference between the standard RNA and the target sequen ce within live r RNA samples is the number of base pairs. The

standard RN A 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

MgCl 2 (from AmpliTaq Gold) 2.24 μl

Vtg1 Standard Forward ( TAATACGACTCACTATAGGGAGA TTCCTCGGATACGGCACAAT) 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 (Th ermo Scientific) following the manufacturer’s instructions. This will provide a conc entration in μg/μl. Calculate the number of cop ies 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 10 23 

### molecules (= copies) Reagent P er 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/ μ l = 1.52 X 10

– 7 

### g/ μ l and a total size of 3,397 bp:

MW = 3,397 bp X 660 daltons/bp = 2.24 X 10 6 

### daltons

1 mol = 2.24 X 10 6 

### g 1 mol = 6 X 10 23 

### 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 sh ould 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 10 10, 

### 10 9 

### , 10 8 

### , 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 10 10 

### ) would

have a concentration of 2.5 X 10 9 

### copies/ μl. For instance, if the standard RNA has a

concentration of 2.3 X 10 11 

### copies/μl then this would be diluted to 2.5 X 10 9 

### copies/μl by adding 91 μl of RNase/DNase free water to every 1 μl of the standard RNA. This 2.5 X

10 9 

### copies/μl standard is called STD10 since when used as prescribed with 4 μl per well,

there is 1.0 X 10 10 

### copies per well. The other standards (STD9 thr ough STD3) are

serial ly diluted from STD10 with the following formula maintained: STD N 

### = 2.5 X 10 N

- 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 quantific ation 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 t he 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 proximi ty 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 int o 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 w ith 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 quali ty 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

Mn 2+ 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 s ample 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 (Appl ied 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 th e baseline range and the critical threshold (C t 

### ) 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 C t 

### 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 C t 

### 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 Med aka). 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

- ti me 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 c onverted 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

### Public Review Draft of December 17, 2014

Page 60 of 60 10 .2.2 Template for Rec ording 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