Chemical Safety      and Pollution Prevention      (7101) EPA No. 740-C-15-002 July 2015

Endocrine Disruptor Screening Program Test Guidelines

OCSPP 890.2200:

Medaka Extended One Generation Reproduction Test (MEOGRT)

NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a).  Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic Substances (OPPTS).  To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline's number.  Any test guidelines developed after April 22, 2010 will use the new acronym (OCSPP) in their title.

The OCSPP test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA and/or FFDCA.  This document provides guidance for conducting the test, and is also used by EPA, the public and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA.  As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice.  At places in this guidance, the Agency uses the word "should."  In this guidance, the use of "should" with regard to an action means that the action is recommended rather than mandatory.  The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.

For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the left side navigation menu.  You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 890.2200:  Medaka Extended One Generation Reproduction Test       (a)  Scope.       (1)  Applicability.  This guideline is intended to be used to help develop data to submit to EPA 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 the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).              (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 determine if a pesticide chemical, or other substances, may pose a risk to human health or the environment due to disruption of the endocrine system. Tier 2 testing is designed to identify any adverse apical effects caused by the substance which may be attributable to possible endocrine interaction, and establish a quantitative relationship between the dose and that adverse effect.  The determination whether to require additional Tier 2 testing is made on a weight-of-evidence basis taking into account data from the Tier 1 assays and other scientifically relevant information available.  The fact that a substance may interact with a hormone system, however, does not necessarily mean that when the substance is used, it will cause adverse effects in humans or ecological systems.  The EPA guidance document "Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing" (Ref. 1) explains this process.

The Medaka Extended One Generation Reproduction Test (MEOGRT) is a two-generation test used to characterize the likelihood, nature, and dose-response relationship of any estrogen-, androgen-, and thyroid-related effects caused by a chemical on fish.  The Japanese medaka (Oryzias latipes) is the appropriate species for use in this test guideline, given its short life-cycle and the possibility to determine its genetic sex (Ref. 2), a critical component of this test guideline.  The specific methods and observational endpoints detailed in this guideline are applicable to Japanese medaka alone.  Chemicals that go through Tier 1 screening and are determined to be bioactive in the estrogen, androgen, and/or thyroid hormone systems may require additional Tier 2 testing to characterize adverse outcomes.  The fact that a substance may interact with a hormone system, however, does not necessarily mean that when the substance is used, it will cause adverse effects in humans or ecological systems.  The decision to require the MEOGRT is based on EPA's weight-of-evidence determination of the Tier 1 screening data and other scientifically relevant information.  Tier 2 tests, such as the MEOGRT, are designed to identify any adverse apical effects which may be caused by endocrine interaction of substance in the relevant taxonomic group, and to establish a quantitative relationship between the dose and that adverse effect.  Additional endpoints are also included in the guidelines that may provide information regarding the general Adverse Outcome Pathway(s) (AOPs) affected by the test substance.  This test guideline is part of the Tier 2 tests included in the OCSPP 890 series for the Endocrine Disruptor Screening Program.         (3) Source.  This OCSPP harmonized test guideline was developed through a process of harmonization with Test Guideline 240 published by the Organization for Economic Cooperation and Development (Ref. 3).		       (b)  Purpose.  This guideline is intended for use in developing data, specifically characterizing any adverse apical effect(s) which may be caused by endocrine interaction of a chemical in fish and establish a quantitative relationship between the dose and that adverse effect(s). The tests include exposure during the most sensitive life stages, and provide the opportunity for identification of dose-response effects.  To fulfill this purpose, the MEOGRT is a longer-term study than the Tier 1 fish short-term reproduction (FSTRA, OCSPP 890.1350; Ref. 4) 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.  For the MEOGRT to be conclusive, a discernible cause-effect relationship needs to exist between exposure to the test substance and an adverse effect.         (c)  Introduction.   This test protocol is intended to measure chemical effects on reproduction and reproductive development in Japanese medaka (O. latipes). 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 (Ref. 5; Ref. 6), OCSPP 850.1500 (Fish Life-Cycle Toxicity Test; Ref. 7), and OECD counterparts (Ref. 8-15).   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 such as fecundity and fertility can be directly linked to adverse population outcomes through population models.  The endpoints (e.g. survival and growth) typically measured in the chronic toxicity tests, the fish full life-cycle test (Ref. 7) and the early life-stage test (OCSPP 850.1400; Ref. 16) are also included in the MEOGRT.  These are used to evaluate the concentration-response of a chemical working through any endocrine or non-endocrine mediated AOP.  The question of whether a test substance has endocrine-mediated effects at lower concentrations than non-endocrine-mediated toxicities cannot be unambiguously addressed by the apical endpoints alone and will rely on other diagnostic biochemical (e.g., vitellogenin induction), histopathological (e.g., Leydig cell hyperplasia) effects, or changes in secondary sexual characteristics (e.g., anal fin papillae count).  Definitions of key terms are provided in Appendix 1.       (d)  General Experimental Design.         (1)  Timeline.  Table 1 summarizes the exposure and measurement endpoint timelines for the MEOGRT.  The 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 exposing reproductively active fish (at least 12 wpf) for the first 21 days of the test during which time the test substance and/or its metabolites are presumed to be distributed to the gametes and tissues of these fish.  At the beginning of the fourth week of exposure, eggs are collected to start the F1 generation and growth measurements are taken of the F0 fish.  Fish in the F1 generation are reared for a total of 15 weeks post fertilization (wpf).  In addition, a subset of the F1 fish is sampled at 9 wpf for evaluation of various endpoints.  Reproduction is assessed in adult F1 fish for 21 continuous days during the period of 12 to14 wpf.  Prior to the humane killing of the F1 adults, eggs are collected and allowed to hatch ending the MEOGRT.   Table 1.  Exposure and measurement endpoint timelines within the MEOGRT.                      MEOGRT Exposure and Endpoint Timeline                                   Generation                                                                               F0                                        1                                        2                                        3                                        4                                                                                Life-stage Key                                                                               F1                                                                                1                                        2                                        3                                        4                                        5                                        6                                        7                                        8                                        9                                       10                                       11                                       12                                       13                                       14                                       15                                                                                Embryo                                                                               F2                                                                                1                                        2                                                                         Eleutheroembryo                                                                           Study Week                                        1                                        2                                        3                                        4                                        5                                        6                                        7                                        8                                        9                                       10                                       11                                       12                                       13                                       14                                       15                                       16                                       17                                       18                                       19                                                                            Juvenile                                                                                Endpoints                                                                            Subadult                                                                            Fecundity                                       F0                                                                                F1                                                                                Adult                                                                            Fertility                                       F0                                                                                F1                                                                                Hatching                                                                                F1                                                                                F2                                         7 groups of replicates *  5 for test substance treatments *  2 for controls (4 if solvent is used) Within-group design *  12 reps for reproduction, pathology, growth, SSC (wks 10-18) *  6 reps for hatching, survival, vtg¸ growth, (wks 1-9)                                                                            Survival                                                                                F1                                                                                F1                                                                                F1                                                                                Growth                                                                                F0                                                                                F1                                                                                F1                                                                                Vitellogenin                                                                                F1                                                                                Secondary sex                                                                                F1                                                                                F1                                                                                Histopathology                                                                                F1                                                                                Study Week                                        1                                        2                                        3                                        4                                        5                                        6                                        7                                        8                                        9                                       10                                       11                                       12                                       13                                       14                                       15                                       16                                       17                                       18                                       19

      (2)  Treatments and Replication.   This guideline recommends five test substance treatments and a negative (dilution water) 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 the F0 generation, each test substance treatment has six replicates while the negative control treatment has 12 replicates.  Solvents are highly discouraged, and if used, a justification for both the use of a solvent and the choice of solvent should be included in the MEOGRT report.  If a solvent is used, two types of controls are necessary: a) a solvent control, and b) a negative control. These two control groups should each consist of a full complement of replicates at all points within the MEOGRT timeline (shown in Table 1).

During the first 9 weeks of the F1 generation, this replicate structure remains the same.  However, for the remainder of the F1 generation, the number of breeding pair replicates per treatment is optimally doubled to 12 replicate pairs and the number of replicates in the negative control is doubled to 24 replicate pairs, and another 24 replicate pairs in the solvent control, if a solvent is used.  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 the F0 generation, daily replicate fecundity (number of spawned eggs), and daily replicate fertility (number of fertile eggs) are recorded for 21 continuous days.  Upon termination of the F0 generation, growth parameters are measured.  In addition, hatching success is recorded for the embryos spawned by F0 generation fish.  At the adult life-stage of F1 generation (15 wpf), 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 are sacrificed for growth, secondary sex characteristics, and histopathology assessment.  At the subadult (9 wpf) life-stage of F1, fish are sampled for growth, secondary sex characteristics, gonad phenotype, and liver vitellogenin (vitellogenin 1; vtg1) mRNA assessment or liver vitellogenin protein assessment.  Survival data are collected for the F1 generation offspring at 4, 9, and 15 wpf. Table 1 (noted above) illustrates the timeline for assessing the endpoints measured in the MEOGRT.  The endpoints are summarized in Table 2.   Table 2.  Endpoint overview of the MEOGRT*  Life-stage Endpoint* Generation Embryo (2 wpf) Hatchability (% and time to hatch) F1, F2 Juvenile (4 wpf) Survival (hatch to 4 wpf) F1

Subadult (9 wpf) Survival (4 to 9  wpf) F1

Growth (length and weight)

Vitellogenin

Secondary sex characteristics (anal fin papillae)

External sex ratio

Time to 1[st] spawn

Adult (12-14 wpf) Reproduction (fecundity and fertility for 21 days) F0, F1

Adult (15 wpf) Survival (from 9 to 15 wpf) F1

Growth (length and weight)

Secondary sex characteristics (anal fin papillae)

Vitellogenin (VTG protein)

Histopathology (gonad, liver, kidney)

*These endpoints are to be statistically analyzed.  	(4)  Genotypic Sex.   All endpoints are analyzed in the context of the genetic sex of the individual fish. Japanese medaka has a XX/XY sex determination system in which the only functional gene identified on the Y chromosome is the dmy gene (Ref. 17). This presence of the dmy gene indicates a XY individual regardless of phenotype (Ref. 18; Ref. 19), which may be altered by exposure to a test chemical.  The dmy gene determination for an individual fish provides a description of the genetic predisposition of the gender phenotype for each fish.  Therefore, the dmy gene status of each fish is essential for proper analysis of biological data.  The dmy gene 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 to properly analyze the subadult data.       (e)  MEOGRT Prologue        (1)  Test Species.  The test species is Japanese medaka, Oryzias latipes, because of its short life cycle and the possibility to determine genetic sex.  Japanese medaka have been used a model fish for studying development and reproduction for over 60 years (e.g., Ref. 20). Many published methods exist for its culture (Ref. 21; Ref. 22; Ref. 23), and data are available from short-term lethality, early life-stage and full life-cycle tests (Ref. 5; Ref. 6; Ref. 24; Ref. 25; Ref. 26). All fish are maintained on a 16 hour light: 8 hour dark photoperiod.  The fish are fed live brine shrimp, Artemia spp., nauplii which may be supplemented with a commercially available flake food if necessary.  Commercially available flake food should be regularly analyzed for contaminants.   As long as appropriate husbandry practices are followed, no specific culturing protocol is required.  For example, medaka can be reared in 2 L tanks with 240 larval fish per tank until 4 wpf, then they can be reared in 2 L tanks with 10 fish per tank until 8 wpf, at which time, they transition to breeding pairs in 2 L tanks.  The exposure phase should be started with sexually dimorphic adult fish from a laboratory supply of reproductively mature animals cultured at 25 +- 2°C.  The fish should be actively spawning prior to the start of the test. The age and weight of the fish selected should be typically more than 12 wpf, but not greater than 16 wpf and have recommended weights of >= 300 mg for females and >= 250 mg for males.  The range in individual weights, by sex, at the start of the test should be kept within +- 20% of the arithmetic mean weight of the same sex.  A subsample of fish should be weighed before the test to estimate the mean weight.       (i)  Selection of Test Fish.  Test fish  (F0 generation) 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 and illumination similar to those used in the test.  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. In addition, each breeding pair of F0 should be genetically verified to be XX  -  XY to avoid the possible inclusion of spontaneous XX males.  Small tissue samples of the tail fin are analyzed for dmy using the same procedures described during the in-life portion of the MEOGRT.  A minimum of 42 breeding pairs (54 breeding pairs if a solvent control is used) are considered necessary to initiate the MEOGRT to ensure adequate replication. In addition, each breeding pair of F0 should be verified to be XX-XY (i.e. normal complement of sex chromosomes in each sex) to avoid the possible inclusion of spontaneous XX males.  It is strongly encouraged that several more breeding pairs than the 42 pair minimum be available for inclusion in the test.  In this way, if pairs stop spawning prior to the start of the test they can be substituted with actively spawning pairs.  If this recommendation is followed, the MEOGRT should be initiated by selecting 42 breeding pairs from all available breeding pairs.  All breeding pairs will have daily fecundity values measured 4 to 7 days prior to the start of the test.  The selected 42 breeding pairs are those in the middle of the distribution of mean fecundities.  The mean daily fecundity of the selected breeding pairs should be greater than 20 eggs/pair/day.  A randomized block design should be used to ensure a balanced distribution of pairs to the treatments based on reproductive performance.       (ii)  Mortalities in Culture Fish.  Mortalities in the culture fish should be recorded and the following criteria applied following a 48 hour settling-down period:          *       Mortalities of greater than 10% of the culture population in seven days preceding transfer to the test system: reject the entire batch;          *       Mortalities of between 5% and 10% of the population in the seven days preceding transfer to the test system: acclimation for seven additional days to the 2 week acclimation period; if more than 5% mortality during the second seven days, reject the entire batch;          *       Mortalities of less than 5% of the population in the seven days preceding transfer to the test system: accept the batch.        Fish should not receive treatment for disease in the two-week acclimation period preceding the test and during the exposure period, and disease treatment should be completely avoided if possible.  Moribund fish or fish with clinical signs of disease should not be used in the study. A record of observations and any prophylactic and therapeutic disease treatments during the culture period preceding the test should be maintained.          (2)  Water.  Any water in which the test species shows suitable long-term survival and growth 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 chemical) or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis.  Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl, SO4), pesticides, total organic carbon and suspended solids should be made, for example, every six months where a dilution water is known to be relatively constant in quality.  The pH of the water should be within the range 6.5 to 8.5, but during a given test it should be within a range of +- 0.5 pH units.    The dilution water alkalinity, hardness, and total organic carbon should be reported.  Some chemical characteristics of acceptable dilution water are listed in Table 3.

            	 Table 3.  Some Chemical Characteristics of Acceptable Dilution Water Substance  Limit concentration  Particulate matter  5 mg/L Total organic carbon  2 mg/L Un-ionized ammonia  1 μg/L Residual chlorine  10 μg/L Total organophosphorous pesticides  50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls  50 ng/L Total organic chlorine  25 ng/L Aluminum  1 μg/L Arsenic  1 μg/L Chromium  1 μg/L Cobalt  1 μg/L Copper  1 μg/L Iron  1 μg/L Lead  1 μg/L Nickel  1 μg/L Zinc  1 μg/L Cadmium  100 ng/L Mercury  100 ng/L Silver  100 ng/L          (3)  Exposure System.  The design and materials used for the exposure system is not prescribed, but certain test conditions are essential to the proper implementation of the MEOGRT. A flow-through diluter system is recommended for this protocol.  A flow-through exposure system is highly recommended to achieve performance criteria. Static renewal systems are strongly discouraged.  The literature describes several different exposure systems used in medaka testing (e.g., Ref. 6; Ref. 24, Ref. 25; Ref. 27; Ref. 28).  For further information on setting up flow-through exposure systems, please refer to the ASTM Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (Ref. 29).  Examples of exposure systems are shown in Appendix 2. A summary of test conditions is presented in Table 4.        (i)  Components.  Glass, stainless steel, or other chemically inert material should be used for construction of the test system that has not been contaminated during previous tests.  The system components including aquaria, dilution cells, pumps, and delivery lines, should have water-contact components of glass, stainless steel, Teflon[(R)], or other inert material.  However, suitable plastics can be utilized if it has been shown that they will not compromise the test.  Exposure tanks should be glass aquaria with a recommended volume of approximately 1.8 L.         (ii)  Water Delivery.  The system is required to support all chemical treatments and treatment controls up to 84 aquaria during reproduction (up to 108 aquaria during reproduction if a solvent is used).  The flow rate to each aquarium should be sufficient to maintain both biological conditions and chemical exposure.  With 1.8 L aquaria, a flow rate of 20 mL/minute usually is sufficient.  Delivery of each treatment to the appropriate aquaria can be accomplished with different systems including appropriately sized peristaltic pumps or metering pumps with manifold systems (see Appendix 2 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 be wide spectrum and should provide a 16 hour light:8 hour dark photoperiod at an intensity range of  approximately 150 lux at the water surface.  If additional light is required during certain test tasks (i.e., collection of eggs during reproduction), increased light intensity may be used, but should be reduced back to the specified range as soon as possible.         (iv) Temperature.  Temperature conditions should be maintained throughout the test (see Table 3).  One option uses low wattage silicon rubber heaters controlled by a digital temperature controller.  This dry system controls tank temperatures as well as water bath systems, and has the advantage of allowing exposure aquaria to be removed from the system without water dripping and allows leaking aquaria to be easily detected.  Regular recording of the temperature of the aquaria should be done.  If a consistently high performing temperature regulation system is in place, a minimum of two replicates in each treatment should be measured daily.  The replicates in each treatment should be rotated so that each replicate is measured approximately the same number of times during the test.  If temperature deviations occur, more frequent measurements of more than two replicates per treatment should be done to adequately record the temperature profile of the bioassay system.        (v)  Curtains.  Medaka can react adversely (e.g., reduced fecundity) by activity outside their aquaria.  Elevated light intensity may also lower fecundity.  To maximize medaka reproduction within the MEOGRT, it is recommended that the test aquaria are visually isolated with curtains (or by other means) from the general laboratory environment.         (4) Test Chemical 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 (Ref. 30).  Test substances which are solid at room temperature and are moderately soluble in water can be introduced using glass wool column saturators (Ref. 30).  Characteristics which indicate that the test substance may be difficult to test in aquatic systems include: high log octanol-water partitioning coefficients (log Kow), high volatility, susceptibility to hydrolysis, and susceptibility to photolysis under ambient laboratory lighting conditions.  Other factors may also be relevant to determining testability and should be determined on a case-by-case basis.  For guidance on testing difficult substances and mixtures in flow-through systems refer to OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Ref. 31) and for conducting aquatic toxicity testing in general, refer to USEPA Guidance Document on Special Considerations for Conducting Aquatic Laboratory Studies.        All efforts should be made to avoid solvents or carriers. If solvent carriers are used, appropriate solvent controls should be evaluated in addition to non-solvent (negative) controls (dilution water only).  In the event that use of a solvent is unavoidable, and microbial activity (bio-filming) occurs, recommend recording/reporting of the bio-filming per tank (at least weekly) throughout the test. Ideally, the solvent concentration should be kept constant in the solvent control and all test treatments. If the concentration of solvent is not kept constant, the highest concentration of solvent in the test treatment should be used in the solvent control.  If a solvent is used, the concentration of the solvent should be kept as low as possible, (e.g., <20 uL/L) to avoid potential effect of the solvent on endpoints measured (Ref. 32).   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 test chemical solutions to be delivered to tanks is critical to the success of a long-term bioassay.  There are several design considerations for a test chemical preparation system.  The preparation of individual test chemical concentrations 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 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 2.       (5)  Test Substance Concentration Selection.  It is recommended to use five chemical concentrations plus control(s).  For the purposes of this test, results from the Tier 1 EDSP studies, in particular, the fish short-term reproduction assay (Ref. 4; Ref. 33), should be used, as well as any other pertinent information, to the extent possible, in determining the highest test concentration so as to avoid concentrations that are overtly toxic.  Prior to running the MEOGRT, a range-finding experiment is recommended.   A range-finding test should be conducted under conditions (water quality, test system, animal loading) similar to those used for the definitive test. As a suggestion, the range-finding test may start with young 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 (e.g.., 2 replicates per treatment and 10 larvae per replicate).  The number of treatments will vary depending on the quality of the information regarding the test substance, but minimally there should be three doses of the test substance plus a control.  While ancillary endpoints can be taken (i.e., growth), the primary data to be collected are for survival.  In addition, including some reproducing adult pairs as another sensitive life stage with egg production as the primary endpoint is also recommended. If use of a solvent is necessary (and no historical data are available), the range-finding test can be used to identify suitability of the solvent.   The results of the range-finding experiment should serve to set the highest test concentration in the MEOGRT.  The data from both these life-stages should be considered and the lower effective concentration of the two studies used to determine a possible high concentration for the test.  Once the highest test concentration is established, the recommended concentration separation (spacing factor) between adjacent test concentrations should generally be no less than 2-fold and no greater than 3.2-fold (half-log).  The lowest concentration should be a factor of 10- to 100-times lower than the highest concentration.           (6)  Analytical Measurement of Test Substance.  After preparation of the test substance stock solution, the concentration in the test system should be made using appropriate methods.  Concentrations in exposure tanks should be measured prior to adding fish to verify that target concentrations are reached. Preceding the initiation of the test exposure it is important to verify that the test substance dilution and delivery system can provide the desired test substance concentrations to the exposure aquariums.  The concentrations of the test substance should be measured in the 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 measured using methods such as spectrophotometry, gas chromatography-electron capture detection (GC-ECD), gas chromatography-mass spectroscopy (GC-MS), HPLC (high performance liquid chromatography) with an appropriate detector, or ion-chromatography with conductivity detection. A summary of test conditions for the test is provided in Table 4.   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 The nominal test temperature is 25 °C.  The mean temperature throughout the test in each tank is 24-26°C. Illumination quality Fluorescent bulbs (wide spectrum, 100-150 lux, ̴150 lumens/m2 )   Photoperiod 16 h light: 8 h dark  Number of test organisms per replicate F0:  2 adults/replicate (aquarium);  F1: initiated with maximum 20 embryos/replicate, 12 hatched larvae (eleutheroembryos)/replicate at hatch, then 2 adults (XX-XY breeding pair) at 9 wpf for reproductive phase.

Example aquarium size 18x9x15 cm  Example aquarium effective volume ~1.8 liters  Volume exchanges of test solutions If using the size aquarium noted above, then use a minimum of 5 volume renewals/day; however, at least 16 volume replacements/day (20 ml/min flow) is preferred.  Age of test organisms at initiation F0: >12 wpf recommended, not to exceed 16 wpf Number of treatments Recommended 6 (5 test substance treatment plus 1 negative control); 7 if a solvent is used Number of. replicates per treatment  Minimum 6 replicates/treatment for test substance treatment; minimum of 12 replicates/control treatment*; replication structure doubled within reproduction phase (F1) for fecundity assessment. See Figure 1. Number of organisms per test A minimum of 84 fish in F0 and 504 fish in F1 will be used.  If a solvent control is needed, then a minimum of 108 fish in F0 and 648 fish in F1 will be used.  The unit counted is the post eleutheroembryos. Aeration None, unless dissolved oxygen concentration approaches<60% of air saturation value. Dilution water Well water, reconstituted water or dechlorinated tap water previously demonstrated to be suitable for medaka Exposure period 19 weeks total (from F0 to F2 hatching) * If solvent is used, then 12 solvent controls as well as 12 negative controls recommended.       (f)  Procedures        (1) Initiation of Test.   Breeding pairs that have met the criteria detailed in Section (e)(1)(i) above should be used to initiate exposure.  It is recommended to begin with 12 replicate breeding pairs in the control treatment.  There should be six replicate breeding pairs in each of the five test substance treatment levels for a total of 42 breeding pairs to start the MEOGRT (54, if a solvent control is needed).  Each breeding pair is randomly assigned a treatment (e.g., T1-T5 and control) and a replicate (e.g., A-L in controls and A-F in treatment), and then placed in the exposure system with the appropriate flow to each replicate aquarium.  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. After spawning, all eggs are collected by carefully removing any attached eggs from the female and by siphoning any eggs from the bottom of each aquarium.  Filaments are removed from all eggs and the eggs from each treatment are pooled.  Fertile, healthy embryonated eggs are then randomly selected for incubation. Using a good quality dissecting microscope, one can see hallmarks of early fertilization/development such as raising of the fertilization membrane (chorion), ongoing cell division, or formation of the blastula.  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 2 for example apparatus) until hatch. It is preferable that the embryos are collected on a single day; however, if there are not enough embryos (120 per test substance treatment; 240 per control group), the embryos may be collected over two days.  If collected over two days (Study Days 22 and 23), all embryos within the treatments that were collected on the first day are pooled with those collected on the second day  then randomly redistributed to each of the treatment replicates.  Each replicate incubator will have 20 embryos per incubator (120 per test substance treatment; 240 per control group).  If a single treatment requires a second day of collection, all treatments (including controls) need to follow this procedure.  If after the second day of collection there are still 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 should 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 should be taken to ensure that the incubators do not become fouled with food which may lead to embryo mortality. To reduce mortality, embryos may be agitated within the incubator, for example by aeration, delivery of the test solution or by moving the egg incubator vertically in the water column.  Incubators should be checked daily for mortalities.  Mortalities are recorded and all dead embryos are removed from the incubators.  If agitation is used, the agitation is stopped on the morning of the first expected day of hatch (e.g., at 25°C, hatching typically begins on the 8 dpf and continues for about 2 more days). For each treatment and control upon hatching, the hatched larvae (e.g., eleutheroembryos) are counted daily, pooled, and distributed to each of the replicate aquaria (Figure 1).  This can be done by randomly selecting an eleutheroembryo from the treatment pool and then sequentially adding it to a replicate aquarium.   Each replicate aquarium should receive 12 eleutheroembryos.  If there are too few eleutheroembryos to fill all replicates, then the number of replicates that are to be loaded is reduced until 12 eleutheroembryos per replicate can be achieved.  Any additional eleutheroembryos are humanely killed with anaesthetic.  Embryos that have not hatched after a prolonged incubation time are considered non-viable and discarded. The day this occurs is defined as twice the median day of hatching in controls (e.g., at a temperature of 25.5 °C, typically this is the 16[th] day post-fertilization).	       (ii) Setup of Breeding Pairs.  Determination of genotypic sex via fin clips is done at 9 wpf (i.e., Test Week 12 for F1 generation).  All fish within a tank are anesthetized and a small tissue sample is taken from either the dorsal or the ventral tip of 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. It is essential that the integrity of the sample identification be maintained and contamination between samples be 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 test data unreliable and potentially impacting the validity of the test.  Once the DNA is extracted, a polymerase chain reaction (PCR) method is used to amplify the dmy gene, if present. As discussed in section (d), General Experimental Design, the presence of the dmy gene is a definitive indication of an XY (male) individual, regardless of phenotype.    The dmy gene information is used to establish XX  -  XY breeding pairs (in both F0 and F1 generation adults) regardless of external phenotype which may be altered by exposure to an endocrine-disrupting chemical.  As a suggested procedure, the fish from a replicate can be housed in small cages, if possible one per cage, in the replicate tank.  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., dorsal vs ventral tip) when taking the tissue sample for dmy gene analysis.  Soon after the genotypic sex of each fish is determined, two XX fish and two XY fish from each replicate are randomly selected for pooling (Figure 1).  After the 12 total XX fish (24 for control(s)) and the 12 total XY fish (24 for control(s)) for each treatment are pooled, fish are randomly selected to produce breeding pairs.  If a replicate does not have either two XX or two XY fish, appropriate fish should be obtained from other replicates within the treatment.  The priority is to have the recommended number of replicate breeding pairs in each treatment group and in each control group.  Please note the following: a) pooling and re-distribution truncates the replicate lineage across life-stages and generations preventing direct intra-replicate statistical comparisons, b) randomly selected XX  -  XY breeding pairs are essential to the validity of the test, and c) fish with obvious abnormalities (swim bladder problems, spinal deformities, extreme size variations, etc.) should be precluded when establishing breeding pairs.   	(iii) Sampling of Subadults (9 wpf).  After the breeding pairs have been established, the fish not selected as breeding pairs are humanely killed for measurement of subadult endpoints.  Unlike the fish selected to become breeding pairs, the replicate identity of each sampled subadult fish is maintained.  Also note that it is essential that the genotypic sex of each fish, that has already been determined, is maintained since all endpoint data are analyzed in the context of the genotypic sex of the specific fish.   Each fish is euthanized and measured for a variety of endpoints including: 9 wpf survival, growth, liver vitellogenin (mRNA copy number or protein concentration), and anal fin papillae number (secondary sexual characters (SSC)).    The liver is dissected for quantification of vitellogenin.  If vitellogenin mRNA is quantified, the 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 be 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 6 for the necropsy procedure, Appendix 7 for counting anal fin papillae, and Appendix 8 and 9 for example protocols for the extraction of RNA and the analysis of vitellogenin. For the purposes of calculating a simple sex ratio, medaka with more than one anal fin papillary processes are defined as a phenotypic males and those with no anal fin papillae are defined as phenotypic females.         (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.  F0 and F1 eggs are collected daily from each breeding pair for 21 consecutive days.  Eggs are gently removed from netted females and siphoned from the bottom of the aquarium each morning.  Both fecundity and fertility are recorded daily for each replicate breeding pair.  Fecundity is defined as the number of eggs spawned, and fertility is functionally defined as the number of fertile and viable eggs at the time of counting.  Counting should be done as soon as possible after egg collection.    For the most part, the process for the collection of eggs and hatch assessment is done in the same manner for the F0 and F1 generations; however, the replication per treatment and per control(s) is different.  In the F1 generation, eggs are collected from 12 replicate breeding pairs per treatment (24 replicates in controls; 48 if a solvent control is needed); whereas, in the F0 generation, there are six replicate breeding pairs per treatment (12 replicates in controls; 24 if a solvent control is needed).  To assess embryo viability and hatch success, all viable embryos are pooled and systematically distributed to six replicate incubators (12 incubators for controls; 24 if a solvent control is needed) as was done in the F0 generation.  If needed, a second day of collection can be used as long as the first and second collection days are pooled as in the F0 generation.  Proceed as described previously in section (f)(2), 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 are considered non-viable and should be discarded appropriately.         (v) Sampling of Adults.  The adults are humanely killed and various endpoints are assessed at 15 wpf (i.e., following Test Week 17).  While the tail is removed and fixed or photographed to assess the number of anal fin papillae (see guidance in Appendix 7), no other tissue dissection is performed to maintain all tissues and organs in their in situ orientations.  The body cavity is opened to allow perfusion with appropriate fixative prior to submersing the entire body in the fixative and each adult fish is evaluated histologically for pathology in the gonads, kidneys and liver tissue as described in the Histopathology Guidance (Appendix 12). Mechanistic endpoints evaluated in this assay (e.g., vitellogenin, SSCs and certain histopathology effects) may be influenced by systemic or other toxicities. Consequently, liver and kidney histopathology is assessed in detail to help better understand any responses in mechanistic endpoints.  "Reading down" from the highest treatment group (compared to the control) to a treatment with no effect may be considered; however, it is recommended that user consult the histopathology guidance in Appendix 12.  The gonad phenotype is also derived from this evaluation.   It is recommended that a tissue sample be taken to repeat the dmy analysis to verify the genetic sex of specific fish when necessary. For instance, it is strongly recommended that if a breeding pair fails to produce more than a minimal number of eggs, the genetic sex of both fish be re-assessed to verify that the pair was indeed XX  -  XY.         (vi) Ongoing Observations.  During the test, observations of behavior should be made at least once daily, and any unusual behavior should be noted.  In addition, any mortality should be recorded and survival to 4 wpf (Test Day 44), from 4 wpf to 9 wpf (Test Day 80), and from 9 wpf to 15 wpf (Test Day 121) should be calculated (see Table 1 and Table 5).  In addition, during the time leading up to the selection of breeding pairs, each replicate should be monitored for its first spawn.  The study day that this occurs should be recorded, but statistical analysis of this data is not performed. 	(3) Detailed Timeline.  A timeline for the MEOGRT was described previously in Table 1.  The MEOGRT includes 4 weeks of exposure to F0 adults and 15 weeks of exposure to the F1 generation.  In week 4 on approximately test day 24, the F1 generation is established and the F0 breeding pairs are humanely killed and weight and length are recorded (Table 1). This is followed by exposure of the F1 generation for 14 more weeks (total of 15 weeks for F1) and the F2 generation for two weeks until hatching. The exposure continues through development and reproduction in the F1 and hatching in the F2 generation.  The total duration of the test is 19 weeks.    An example of a day-by-day schedule for conducting the test is provided as guidance in Appendix 3 and as a Microsoft(R) Excel spreadsheet  as a separate electronic file on the EPA EDSP website: http://www.epa.gov/endo/.   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.   A detailed week-by-week timeline for the test is presented below in Table 5.   Table 5.  Detailed Weekly Timeline for the MEOGRT. Study Weeks 1-3 (F0) 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 There are a total of 42 breeding pairs (54 breeding pairs if a solvent control is needed).  Spawned eggs are collected, counted and assessed for fertility for 21 consecutive days starting at Study Day 1.  Study Week 4 (F0 and F1) On Study Day 22, eggs are collected from each aquarium and each female, pooled by treatment, and systematically distributed to suitable incubation vessels (6 per treatment and 12 for controls) as detailed in (f)-(2)-(iv).  Again, the vessels may be placed in separate "incubator aquaria" set up for each treatment or in the replicate aquarium that upon hatch will contain the eleutheroembryos.

Ideally, all embryos used to start F1 should be collected on the same day; however, a second day of collection may be necessary if an insufficient number of viable embryos are collected on the first day.  If a second day of collection (Study Day 23) is needed, all embryos from both days should be pooled and then systematically redistributed to each of the treatment replicates at 20 embryos per incubator. Mortalities are recorded daily.    Note: If a single treatment requires a second day of collection, all treatments (including controls) need to follow this procedure.  If after the second day of collection there are inadequate numbers of embryos within a treatment to load 20 embryos per incubator, then 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, then reduce the number of replicate incubators until there are enough embryos for 15 per incubator. Additionally, more breeding pairs per treatment and controls could be added in F0 to produce more eggs to reach the recommended 20 per replicate. On Test Day 24, the F0 breeding pairs are humanely killed, and weight and length are recorded. If necessary F0 breeding pairs maybe kept for an additional 1-2 days in order to restart F1. Study Weeks 5-6 (F1) One day before the anticipated start of hatching, stop or reduce the agitation of the incubating embryos to expedite hatching. As embryos hatch on each day, eleutheroembryos are pooled by treatment and then systematically distributed to each replicate aquarium within a specific treatment with no more than 12 eleutheroembryos placed into each aquarium.  This is done by randomly selecting eleutheroembryos and placing a single eleutheroembryo in successive replicates in an indiscriminate draw, moving in order through the specific treatment replicates until all replicates within the treatment have 12 eleutheroembryos.  If there are not enough eleutheroembryos to fill all replicates then ensure as many replicates as possible have 12 eleutheroembryos to start the F1 phase of the test.  The appropriate data are collected to calculate the hatching success of each replicate incubator.  As detailed previously, embryos that have not hatched by twice the median control day of hatch) are considered non-viable and appropriately discarded.  The number of eleutheroembryos are recorded and hatching success is calculated in each replicate. Study Weeks 6-11 (F1)   On Study Day 44, survival of the juvenile fish to this point is recorded as the number per replicate out of the initial number of eleutheroembryos, nominally 12.  The exposure continues as the juvenile fish develop into subadults. Study Week 12.  (F1) 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 genotpyic data for all the remaining subadult samples is retained to ensure that all endpoint data can be related to the genetic sex of each individual fish.  On Study Days 80 and 81 (within three days), after the genotypic sex of each fish is determined, 12 breeding pairs per treatment and 24 breeding pairs for controls are randomly established as detailed in (f)-(2)-(ii). The remaining fish (maximum 8 fish per replicate) are humanely killed and are sampled for the various subadult endpoints as described in (f)-(2)-(vi).  It is essential that the dmy gene status (XX or XY) for all the subadult samples are retained to ensure that all endpoint data can be related to the genetic sex See Appendices 3 and 4 for detailed protocols on DNA extraction and dmy gene analysis.  Study Weeks 13-14. (F1) The exposure continues as the subadult breeding pairs develop into adults.  Study Weeks 15-17. (F1) 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 (Study Days 99-119) in each replicate, and assessed for fecundity and fertility.  Study Week 18.  On Study Day 120, eggs are collected, pooled from each treatment, and systematically distributed to suitable incubation vessels (6 per treatment and 12 for controls; plus an additional 12 controls if a solvent control is used) with no more than 20 embryos per replicate (120 embryos for each test substance treatment and 240 embryos 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 embryo collection, the F1 breeding pairs are terminated humanely killed and analyzed for the various adult endpoints as described in (f)-(2)-(vi). If necessary F1 breeding pairs maybe kept for an additional 1-2 days in order to restart F2. Study Week 19. (F2) One day before the anticipated start of hatching, stop or reduce the agitation of the incubating eggs to expedite hatching. As embryos hatch on each day, eleutheroembryos are pooled by treatment and then systematically distributed to each replicate aquarium within a specific treatment with no more than 12 eleutheroembryos placed into each aquarium.  This is done by randomly selecting eleutheroembryos and placing a single eleutheroembryo in successive replicates in an indiscriminate draw, moving in order through the specific treatment replicates until all replicates within the treatment have 12 eleutheroembryos.  If there are not enough eleutheroembryos to fill all replicates then ensure as many replicates as possible have 12 eleutheroembryos to start the F1 phase of the test.  The appropriate data are collected to calculate the hatching success of each replicate incubator.  As detailed previously, embryos that have not hatched by twice the median control day of hatch) are considered non-viable and appropriately discarded.                                                                                Figure 1.  Pooling strategy used during the MEOGRT.  The figure represents one treatment or (1/2) of a control.  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. Fish can be fed brine shrimp Artemia spp., supplemented with a commercially available flake food if necessary.  Food should be regularly analysed for contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs).  Food with an elevated level of endocrine active substances (i.e., phytoestrogens) that could compromise the response of the test should be avoided.The following feeding schedule is recommended to ensure adequate growth and development to support robust reproduction. Deviations from this feeding schedule may be acceptable, but they should be tested to verify that acceptable growth and reproduction are observed. In order to follow the suggested feeding schedule, the dry weight of brine shrimp per volume of brine shrimp slurry needs to be determined prior to starting the test. This can be done by weighing a defined volume of brine shrimp slurry that has been dried for 24 hours at 60°C on pre-weighed pans. To account for the weight of the salts in the slurry, an identical volume of the same salt solution used in the slurry should 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 6 from dry weight of brine shrimp to volume of brine shrimp slurry to be fed per fish. In addition, weekly aliquots of the brine shrimp slurry should be weighed to verify the correct dry weight of brine shrimp is being fed.        Table 6.  Example Feeding Schedule.	                                      Time                                  (post-hatch)                                  Brine Shrimp                            (mg dry weight/fish/day)                                      Time                                  (post-hatch)                                  Brine Shrimp                            (mg dry weight/fish/day) Day 1 0.5 Day 12 4.2 Day 2 0.5 Day 13 4.5 Day 3 0.6 Day 14 4.8 Day 4 0.7 Day 15 5.2 Day 5 0.8 Day 16-21 5.6 Day 6 1.0 Week 4 7.7 Day 7 1.3 Week 5 9.0 Day 8 1.7 Week 6 11.0 Day 9 2.2 Week 7 13.5 Day 10 2.8 Week 8-sacrifice 22.5 Day 11 3.5 -- --

      (4)  Analytical Chemistry Schedule.  After the exposure is initiated, it is important to monitor the concentration of the test substance in the exposure aquariums frequently enough to allow accurate characterization of the treatment concentrations, and to determine whether the dilution/exposure system is performing as desired.  If it is not, then the appropriate adjustments can be made to mitigate excursions of the test substance concentrations in each treatment as necessary. Weekly sampling of at least two replicate aquaria from each treatment is usually sufficient to define the concentration of each treatment through time.  One of the samples should be taken from the same treatment replicate at each sampling time and the other sample should be selected systematically from one the other possible treatment replicates.  This sampling strategy will provide estimates of test substance concentration in each treatment through time as well as estimates of the within-treatment concentration variance.   It is also important to evaluate other possible sources of variance in the analytical chemistry process by using appropriate measurement replication and chemical standards.  For test substances that appear to be unstable or are subject to biological degradation in the dilution/exposure system, it may be necessary to measure treatments more than once per week.         (5)  System Cleaning Schedule.  The chemical dilution and exposure systems used for the MEOGRT are in contact with water and diluted chemical for a relatively long period. Biofilms invariably form on most wetted surfaces.   The microbiological community that establishes itself will often be able to metabolize the compound being assessed.  Often, detectable decreases in test substance concentration can be observed within three weeks of the start of the assay.  Routine cleaning and disinfection of all wetted components in the diluter/delivery system, and exposure aquaria is recommended approximately every three weeks. A suggested cleaning protocol would be to divert the exposure delivery lines from each replicate aquarium.  The replicate aquaria can usually be left static without flow for the necessary time.  The system components that can be removed should be cleaned with detergent and scrubbed with an abrasive pad.   The delivery lines should be sanitized using a cold sterilant product (e.g., Minncare(R) Cold Sterilant).  As a precautionary note, diluter systems are designed to the particular needs of each user.  The materials of construction may not be compatible with a specific sterilant.  Before disinfecting any dilution system, an evaluation as to whether the system is compatible with the sterilant solution used needs to be done. Analysis of chemical concentrations should be performed both before and after any cleaning of the exposure system.         (6)  Environmental Conditions.  Water quality characteristics should be measured regularly across replicates and treatments to document adequate water quality to sustain healthy medaka.  Dissolved oxygen, and pH should be measured in two replicates in each treatment at least once a week.  Temperature should be monitored continuously and recorded daily. The measured replicates should be rotated within each treatment so that all replicates are measured roughly the same number of times over the test.  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 ug/L.  The dilution water alkalinity, hardness, and total organic carbon should be documented at least once during the test.       (7)  Randomization. At all stages and in most, if not all tasks, it is important to include randomization.  This includes, but is not necessarily limited to, the following procedures:       *       The order in which food is delivered to the aquaria should be randomized to mitigate potential changes in food quantity or quality during the course of feeding.       *       It is essential to randomize the selection of fish when distributing them into the replicates as eleutheroembryos, and designating them to either become part of breeding pairs or be sampled as subadults.       *       When fish are humanely killed either as adults or subadults, the order in which the replicate aquaria are sampled should be random.

      (g)  Performance Criteria and Test Acceptability/Validity.         (1) 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 test.  However, failure in several criteria or failure to meet the fecundity criterion could result in the rejection of the test. When deviations from the test validity criteria are observed, the consequences should be considered in relation to the reliability of the test results, and these deviations and considerations should be included in the test report.  The performance criteria are generally expected for an acceptable test are summarized in Table 7.

       Table 7. Performance criteria for the MEOGRT. Parameter Criteria Test concentrations  Maintain concentrations of the test chemical in solution within +-20% of the mean measured values over the entire test period. Dissolved oxygen concentration >= 60% of air saturation value throughout the test pH pH should be maintained between 6.5-8.5. The inter-replicate/inter-treatment differentials should not exceed 0.5.  Mean water temperature Between 24 and 26°C over the duration of study.  Brief excursions 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. Treatments within the test should not be statistically different from each other.  Mortality in controls  <= 20% mortality in each replicate in the controls. Hatchability of Eggs >= 80% in the controls (in each of the F1 and F2 generations).   Survival after hatching Should be >= 80% (average) until 3 wpf in F1 controls. Should be from >= 90% (average) from 3 wpf through termination in F1 controls.  Mortalities due to technical errors (handling) should not be included in these analyses.   Fish weight at subadult sampling F1 control XX and XY fish mean weight should be >100 mg and mean length >20mm. Growth of substantially less than these values suggests the fish will not achieve sufficient size to spawn with acceptable fecundity. Occurrence of intersex fish XX males should be <5% and XY intersex should be <2% (determined by comparison of genotype and histological sex) in F1 controls. Fecundity Should be >20 eggs/pair/day in F0 and F1 controls. For adequate statistical power, 16 of 24 control pairs (in F1) should produce > 20 eggs/pair/day. Fertility Should be >80% for all eggs produced in F0 and F1 controls (i.e., F1 and F2 eggs) during the assessment.

(2)  Test Validity.  Achieving the following goals will likely deem a test to be considered acceptable/valid:

                                 *       At least three treatment levels with three uncompromised replicates are available for analysis. Excessive mortality, which compromises a treatment, is defined as >20% mortality in 2 or more replicates that cannot be explained by technical error.                                  *       There should be sufficient reproduction in at least the third highest group and all lower groups of the F0 to fill the hatching incubators.                                  *       There should be adequate survival in the third highest test exposure and lower exposure groups in F1 to allow endpoint evaluation at the subadult sampling.                                  *       Survival in the second highest exposure group of the F1 should be >20%.                                  *       Note signs of overt toxicity. Signs of overt toxicity may include, but are not limited to, floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, and being nonresponsive to stimuli.                                  *       A NOEC and LOEC should be determined.

      (h)  Data Analysis. To expedite data reporting and statistical analysis, it is recommended that common templates for data reporting be used.  These templates take the form of Excel spreadsheets that can easily be converted into the CSV file format which is required by the Agency provided statistical tool.  Example formats are provided in Appendix 11 and electronic versions of all templates can be accessed on the EPA EDSP website, http://www.epa.gov/endo.       (1)  Statistical Analysis.  The types of biological data generated in the test 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. Data for continuous endpoints should first be checked for monotonicity by rank transforming the data, fitting to an ANOVA model and comparing linear and quadratic contrasts. Additionally, the issue of using a one-tailed statistical test versus a two-tailed statistical test should be considered.  Unless there is a biological reasoning that would make a one-tailed test inappropriate, it is suggested that one-tailed tests be used.   While certain statistical tests are recommended, if more appropriate and/or powerful statistical methods are developed for application to the specific data generated in the test, those statistical tests could be used to leverage those advantages. The test data should be analysed separately for each genotypic sex.  Failure to do this will greatly reduce the statistical power of any analysis.        (i)  Histopathology data. Histopathology data are reported as severity scores which are evaluated using a newly developed statistical procedure, the Rao-Scott Cochran-Armitage by Slices (RSCABS; Ref. 35). 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 retains test-replication information; the by-slices procedure incorporates the biological expectation that severity of effect tends to increase with increasing doses or concentrations, while retaining the individual subject scores and revealing the severity of any effect found. The RSCABS procedure not only determines which treatments are statistically different (i.e., have higher prevalence of pathology than controls), but it also determines at which severity score the difference occurs providing much needed context to the analysis.   "Reading down" from the highest treatment group (compared to the control) to a treatment with no effect may be considered; however, it is recommended that user consult the histopathology guidance in Appendix 12.  Typically all samples are processed/sectioned after which are read by the pathologist. If using a "read-down" approach, it is noted that the Rao-Scott Cochrane-Armitage by Slices (RSCABS) procedure uses the expectation that as dose levels increase the biological impact (the pathology) will increase as well.  Therefore, one will lose power if only looking at a single high dose without any intermediate doses.  If statistical analysis is not necessary to determine that the high dose has no effect, then this approach may be acceptable.         (ii) Time to event data.  Time to hatch and time to first spawn should be treated as time to event data, with individual embryos not hatching in the defined period or replicates never spawning treated as right-censored data.  Time to hatch should be calculated from the median day of hatch of each replicate.  These endpoints should be analyzed using a mixed-effects Cox proportional hazard model.       (iii) Fecundity data.  The preferred analyses of fecundity examines the overall impact on fecundity for the 21 day observation period.  The raw data are recorded and presented in the study report as the fecundity (number of eggs) per replicate for each day.  The replicate mean of the raw data should be calculated then a square root transformation applied.  A one-way ANOVA on the transformed replicate means should be calculated followed by Dunnett contrasts.  Alternatively, analyses for fecundity data consist of a step-down Jonckheere-Terpstra or Williams' test to determine treatment effects, provided the data are consistent with a monotone concentration-response. With a step-down test, all comparisons are done at the 0.05 significance level and no adjustment for the number of comparisons made. The data are expected to be consistent with a monotone concentration response, which can be verified by constructing linear and quadratic contrasts of treatment means after a rank-order transform of the data. Unless the quadratic contrast is significant and the linear contrast is not significant, the trend test is done. Otherwise, Dunnett contrast is used to determine treatment effects if the data are normally distributed with homogeneous variances. If those requirements are not met, then Dunn's test with a Bonferonni-Holm adjustment is used. All indicated tests are done independently of any overall F- or Kruskal-Wallis test. Further details are provided in OECD 2006. If an understanding of time-by-treatment effects are desired to augment the primary fecundity statistical results described above, the ANOVA model is given by Y= Treatment + Time + Time*Treatment, with random effects of Replicate (Treatment), and Time*Replicate (Treatment).  Here Time refers to the frequency of egg counts (e.g., Day or Week). This is a repeated measures analysis, with the correlations between observations on the same replicates accounting for the repeated measures nature of the data.     Main effects of treatment are tested using the Dunnett (or Dunnett-Hsu) contrasts, which adjusts for the number of comparisons.  Adjustments for the main effect of time are not needed, for with this factor there is no "control" level and every pair of levels is a comparison of possible interest.  For this main effect, if the F-test for the main effect is significant at the 0.05 level, then the pairwise comparisons across levels of that factor can then be tested at the 0.05 level without further adjustment.

The model includes two-factor interactions, so that a main effect for say time, may not be significant even though time has a significant impact on results. Thus, if a two-factor interaction involving time is significant at the 0.05 level, then one can accept the comparisons of levels of time at the 0.05 significance level without further adjustment.       Next are F-tests for significance of treatment within time, the so-called slices in the ANOVA table. If, for example, the slice for treatment and time 12, is significant at the 0.05 level, then the pairwise comparisons for treatment and time 12 can be accepted at the 0.05 level without further adjustment. Finally, for comparisons not falling under any of the above categories, comparisons should be adjusted using the Bonferroni-Holm adjustment to p-values. Further information on analyses of such models can be found in (Ref. 37) and (Ref. 38).

      (iv) All other biological data.  The first formal check of the data is to test whether it violates the assumptions of monotonicity by using linear and quadratic contrasts. If the quadratic contrast is significant and the linear contrast is not, the data are considered non-monotonic. If the data are monotonic, a Jonckheere-Terpstra on replicate medians trend test (as advised in Ref. 36) is recommended.  If the data are non-monotonic, in particular because of the reduced response of the highest one or two treatments, consideration should be given to censoring the dataset so that the analysis is done without those treatments.  This decision will need to be made with professional judgment and all available data, especially data that indicates overt toxicity at those treatment levels.

For weight and length, no transforms are recommended although they may occasionally be necessary.  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 proportion hatching, percent survival, sex ratio, and percent fertile eggs.  The biological data from adult samples has one measurement per replicate, that is, there are one XX fish and one XY fish per replicate aquarium.  Therefore, it is recommended that a one-way ANOVA be done on the replicate means.  If the assumptions of the ANOVA (normality and variance homogeneity as assessed on the residuals of the ANOVA by Shapiro-Wilks test and Levene's test, respectively) are met, Dunnett contrasts should be used to determine treatments that were different from the control.  On the other hand, if the assumptions of the ANOVA are not met, then a Dunn's test should be done to determine which treatments were different from control.  A similar procedure is recommended for data that are 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 numbers of individuals that contribute to the replicate mean for each genotypic sex.  Therefore, it is recommended that a mixed effects ANOVA model be used followed by Dunnett contrasts, if the normality and variance homogeneity assumptions were met (on the residuals of the mixed effects ANOVA).  If they were not met, then a Dunn's test should be done to determine which treatments were different than control.                                         Figure 2.  Flow chart for the recommended statistical procedures for MEOGRT data analysis.         (2)  Special Data Analysis Considerations.       (i)  Use of Compromised Treatment Levels.  Several factors are considered when determining whether a replicate or entire treatment demonstrates overt toxicity and should be removed from analysis.  Overt toxicity is defined as >4 mortalities in any replicate between 3 wpf and 9 wpf that can only be explained by toxicity rather than technical error.  Other signs of overt toxicity include hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and any clinical signs of disease.  For sub-lethal signs of toxicity such as reduced growth and pathology of non-gonadal tissue, qualitative evaluations (rather than statistical) may be necessary, and should always be made in reference to the dilution water control group (clean water only).   If overt toxicity is evident in the highest treatment(s), it is recommended that those treatments be censored from the analysis.         (ii)  Solvent controls.  A solvent should only be considered as a last resort when all other chemical delivery options have been considered.  If a solvent is used, then a dilution water control should be run in concert and a justification for the use of a solvent and the choice of solvent should be included in the test report.   It is recommended that solvent concentration are as low as possible (e.g, <20 μl/L) to avoid potential effect of the solvent on endpoints measured (Ref. 32) and solvent concentrations should not exceed 100 μl/L or 100 mg/L (Ref. 33). In addition, when solvents are used, tanks should be checked for possible microbial activity (bio-filming) should be checked throughout the test. At the termination of the test, the solvent control group should be statistically compared to the dilution water control group for potential effects growth determinants (weight).  If statistically significant differences are detected in these endpoints between the dilution water control and solvent control groups, best professional judgment should be used to determine if the validity of the test is compromised.  If the two controls differ, the treatments exposed to the chemical should be compared to the solvent control unless it is known that comparison to the dilution water control is preferred. If there is no statistically significant difference between the two control groups it is recommended that the treatments exposed to the test chemical are compared with the pooled (solvent and dilution-water control groups), unless it is known that comparison to either the dilution-water or solvent control group only is preferred.       (i)  Data Reporting.  The test report should, at minimum, include the following information:        (1)  Test Substance. Chemical source (and Lot number), chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity (and analytical method for quantification), chemical identity of impurities as appropriate and practically feasible, etc. (including the organic carbon content, if appropriate),  For multi-constituent substance, (e.g., UVBCs and mixtures) characterized as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents.       (2)  Test species. At a minimum, the supplier, available culture history including specific strain information, and any pretreatment should be reported.       (3)  Test conditions.  Test procedure used (test type, loading rate, stocking density, etc.); method of preparation of stock solutions and flow-rate; nominal test substance concentrations, means of the measured values and standard deviations in test tanks and method by which these were attained and evidence that the measurements refer to the concentrations of test substance in true solution;  dilution water characteristics (including pH, hardness, alkalinity, temperature, dissolved oxygen concentration, residual chlorine levels, total organic carbon, suspended solids, and any other measurements made); water quality within test tanks: pH, hardness, temperature, and dissolved oxygen concentration; detailed information on feeding (e.g. brine shrimp source, analyses for relevant contaminants if necessary, e.g., PCBs, PAHs and organochlorine pesticides, any deviations from the feeding protocol and rationale for those deviations), source and treatment of dilution water, average and ranges of water chemistry parameters, light intensity, and tank dimensions. Test procedure used (test type, loading rate, stocking density, etc.).       (4)  Results.   Evidence that controls met the performance criteria; analytical techniques used, statistics, treatment of data and justification of techniques used; tabulated data preferably using the suggested data template for MEOGRT (Appendix 11); detailed report on histopathology including tabulated data using suggested pathology spreadsheet (Appendix 11); results of the statistical analysis preferably in tabular and graphical form; incidence of any unusual reactions by the fish and any visible effects produced by the test substance; mean, standard deviation, and range for each test endpoint; no observed effect concentration (NOEC) for each response assessed; lowest observed effect concentration (LOEC) for each response assessed (at p = 0.05); deviation from the test guidelines and deviations from the performance criteria, and considerations of potential consequences on the outcome of the test.         (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 with putative endocrine-mediated pathways of a test substance occur in medaka, and to quantitatively evaluate those apical effects.  In addition, the MEOGRT should include exposure during the most sensitive life stages, and provide the opportunity for identification 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 provide information both on important apical endpoints that are relevant at the population level, and on endpoints that characterize the AOPs that might be affected by exposure to the test agent.   The test should define a NOEC and have at least one concentration without a statistically significant effect on any of the observed endpoints. It is important to note, however, that if a given exposure level results in substantial mortality or other overt signs of toxicity, responses in other endpoints may be due to general toxicity, and not necessarily mediated via a primary interaction with the endocrine system.  Any lower treatment level(s) should be examined for effects outside of the range of general toxicity.  If all test substance concentrations exhibit mortality or effects on apical endpoint such as growth, fecundity, sex reversal, and intersex, then the assay would need to be repeated with lower concentrations before inferences about possible endocrine activity could be made.        (k)  References.           1) US EPA. 2011.  Endocrine Disruptor Screening Program.  Weight-of-Evidence: Evaluating Results of the EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing.  US EPA, Washington, DC.    2) Padilla S, Cowden J, Hinton DE, Yuen B, Law S, Kullman SW, Johnson R, Hardman RC, Flynn K, Au DWT, 2009. Use of medaka in toxicity testing. Current Protocols in Toxicology 39: 1-36.    3) OECD. 2015. Medaka Extended One Generation Reproduction Test (MEOGRT). Guidelines for the Testing of Chemicals No. 240. OECD, Paris.    4) US EPA. 2009.  Endocrine Disruptor Screening Program.  OCSPP 890.1350: Fish Short-Term Reproduction Assay (FSTRA). US EPA, Washington, DC.    5) Seki M., Yokota H., Matsubara H., Maeda M., Tadokoro H., Kobayashi K. 2003. Fish full life-cycle testing for the weak estrogen 4-tert-pentylphenol on medaka (Oryzias latipes). Environ Toxicol Chem 22:1487-1496.    6) Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H, Honjo T, Kobayashi K. 2001. Life-cycle toxicity of 4-nonylphenol to medaka (Oryzias latipes). Environ Toxicol Chem 20:2552-2560    7) US EPA. 1996. Ecological Effects Test Guidelines. OPPTS 850.1500: Fish life cycle toxicity.  US EPA, Washington, DC    8) OECD. 1998. Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages. OECD Guidelines for the Testing of Chemicals No. 212. OECD, Paris.    9) OECD. 2009. 21-day Fish Assay: A Short-Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition. OECD Guidelines for the Testing of Chemicals No. 230. OECD, Paris.    10) OECD. 2010. Guidance document on the diagnosis of endocrine-related histopathology in fish gonads. OECD Environment, Health and Safety Publications. Series on testing and assessment No.123. OECD, Paris.    11) OECD. 2011. Fish Sexual Development Test. OECD Guidelines for the Testing of Chemicals No. 234. OECD, Paris.    12) OECD. 2012a. Fish Toxicity Testing Framework, OECD Environment, Health and Safety Publications. Series on testing and assessment No. 171. OECD, Paris.    13) OECD. 2012b. Guidance Document on Standardised Test Guidelines for Evaluating Endocrine Disrupters. OECD Environment, Health and Safety Publications. Series on testing and assessment No. 150. OECD, Paris.    14) OECD. 2013a. Fish, Early-life Stage Toxicity Test. OECD Guidelines for the Testing of Chemicals No. 210. OECD, Paris.     15) OECD. 2013b. Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals No. 236. OECD, Paris.    16) US EPA. 1996. Ecological Effects Test Guidelines.  OPPT 850.1400: Fish Early-Life Stage Toxicity Test.  US EPA, Washington, DC.    17) 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.    18) Nanda I, Hornung U, Kondo M, Schmid M, Schartl M. 2003. Common spontaneous sex-reversed XX males of the medaka Oryzias latipes. Genetics 163: 245 - 251.    19) Shinomiya, A, Otake H. Togashi K. Hamaguchi S. Sakaizumi M. 2004, Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations, Zoological Science 21: 613-619.    20) Matsui, K. 1949. Illustration of the normal course of development in the fish, Oryzias latipes. Jpn. Exp. Morph. 5, 33-42.    21) 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.    22) Koger CS, Teh SJ, Hinton DE. 1999. Variations of light and temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes). Biology of Reproduction 61: 1287-1293.    23) Kinoshita M, Murata K, Naruse K, Tanaka M. 2009. Medaka: Biology, Management, and Experimental Protocols, Wiley- Blackwell.    24) Yokota H., Tsuruda Y., Maeda M., Oshima Y., Tadokoro H., Nakazono A., Honjo T., Kobayashi K. 2000. Effect of bisphenol A on the early life stage in Japanese medaka (Oryzias latipes). Environ Toxicol Chem 19:1925-1930.    25) 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    26) 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.    27) 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.     28) Kang I.J., Yokota H., Oshima Y., Tsuruda Y., Yamaguchi T., Maeda M., Imada N., Tadokoro H., Honjo T. 2002. Effects of 17-estradiol on the reproduction of Japanese medaka (Oryzias latipes). Chemosphere 47:71 - 80.    29) ASTM, 2002 ASTM Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.    30) Kahl M.D., Russom C.L., DeFoe D.L., Hammermeister D.E. 1999. Saturation units for use in aquatic bioassays. Chemosphere 39:539-551.    31) OECD. 2002. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications. Series on testing and assessment No. 23. OECD, Paris.    32) Hutchinson TH., Shillabeer N., Winter MJ, Pickford DB. 2006. Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic Toxicology 76:.69 - 92.    33) OECD. 2012c. Fish Short Term Reproduction Assay. OECD Guidelines for the Testing of Chemicals No. 229. OECD, Paris.    34) Green J.W., Springer T.A., Saulnier A.N., Swintek J.  2014. Statistical analysis of histopathology endpoints. Environ Toxicol Chem 33:1108-1116.    35) 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.    36) Hocking, RR. 1985. Analysis of linear models.  Brooks/Cole Publishing Company.  400 pg.      37) Hochberg, Y Tamhane, AC. 1987. Multiple Comparison Procedures, New York: Wiley and Sons.

(l)  List of MEOGRT Appendices.			Page

         (1) Definitions.........................................................................	34          (2) Example Infrastructure.............................................................	35          (3) Example Daily Calendar..........................................................	42          (4) DNA Extraction from Fin Clip Tissue.........................................	46          (5) Example Real-Time PCR Protocol for dmy Gene Analysis in Japanese Medaka..............................................................................	47          (6) Subadult Sampling  -  Necropsy...................................................	52          (7) Counting Anal Fin Papillae......................................................		54          (8) RNA Extraction from Medaka Liver............................................	55          (9) Preparation of RNA for Standard Curve Used during VTG1 QPCR.......	56          (10) QPCR to Quantify Vtg1 mRNA in Liver.......................................	61          (11) Data Reporting Templates.......................................................	64          (12) Histopathology Guidance.......................................................	66

APPENDIX 1: Definitions. ELISA: Enzyme-Linked Immunosorbent Assay Fecundity = number of eggs;  Fertility = number of viable eggs/fecundity;  Fork length (FL): refers to the length from the tip of the snout to the end of the middle caudal fin rays and is used in fishes in which it is difficult to tell where the vertebral column ends www.fishbase.org  Hatchability = hatchlings/number of embryos loaded into an incubator IACUC: Institutional Animal Care and Use Committee Standard length (SL): refers to the length of a fish measured from the tip of the snout to the posterior end of the last vertebra or to the posterior end of the midlateral portion of the hypural plate. Simply put, this measurement excludes the length of the caudal fin. (www.fishbase.org)  Total length (TL): refers to the length from the tip of the snout to the tip of the longer lobe of the caudal fin, usually measured with the lobes compressed along the midline. It is a straight-line measure, not measured over the curve of the body (www.fishbase.org)

Figure 1: Description of the different lengths, used  ECx: (Effect concentration for x% effect) is the concentration that causes an x% of an effect on test organisms within a given exposure period when compared with a control. For example, an EC50 is a concentration estimated to cause an effect on a test end point in 50% of an exposed population over a defined exposure period.  Flow-through test: is a test with continued flow of test solutions through the test system during the duration of exposure. HPG axis: hypothalamic-pituitary-gonadal axis IUPAC: International Union of Pure and Applied Chemistry.  Loading rate: the wet weight of fish per volume of water. Lowest observed effect concentration (LOEC) is the lowest tested concentration of a test chemical at which the chemical is observed to have a statistically significant effect (at p < 0.05) when compared with the control. However, all test concentrations above the LOEC should have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation should be given for how the LOEC (and hence the NOEC) has been selected. Annexes 5 and 6 provide guidance.  Median Lethal Concentration (LC50): is the concentration of a test chemical that is estimated to be lethal to 50% of the test organisms within the test duration. No observed effect concentration (NOEC) is the test concentration immediately below the LOEC, which when compared with the control, has no statistically significant effect (p < 0.05), within a stated exposure period. SMILES: Simplified Molecular Input Line Entry Specification. Stocking density: is the number of fish per volume of water. UVCB: substances of unknown or variable composition, complex reaction products or biological materials. VTG: vitellogenin is a phospholipoglycoprotein precursor to egg yolk protein that normally occurs in sexually active females of all oviparous species. WPF: weeks post fertilization.

APPENDIX 2:  Example Infrastructure.

(a)  Background.  This appendix illustrates examples of infrastructural components that have been successfully used to conduct this test protocol.

(b)  Egg Incubators.  The smooth transition from the adults of a generation to the larvae of the next generation is critical to the success of the assay.  The embryos require careful handling during this period.  The design of an embryo incubator needs to address both husbandry and exposure concerns.  Careless embryo husbandry can result in mortalities and lost embryos resulting in either insufficient eleutheroembryos to continue to the next generation or artificially low numbers for hatching.  Furthermore, embryo development is an important exposure window in which the treatment chemical concentration should remain stable.  Therefore the incubator design should allow for both continuous renewal of the test chemical and easy access to collect water samples from inside the incubator in order to determine the chemical concentration within the incubator. Easy and clean removal of the incubator from the exposure system is also a requirement in order to observe the embryos under magnification, to remove non-viable embryos and to remove eleutheroembryos.  Elements critical to embryo hatching success are: monitoring of the embryos and immediately removing ones that are non-viable; precision handling during transfers between containers; and precise control of air and/or  water flow in the incubator.

The following are short descriptions and illustrations of incubators that have been used to successfully hatch medaka embryos.

   (1)  Flow Though Incubator Tube.

          (a)  Pyrex No. 8240-100 tube			(b) Tank

Figure 1.  Flow Though Incubator Tube.  (a) This type of incubator is constructed from a Pyrex No. 8240-100 tube, cut to a length of 14 cm with slots cut for overflow. (b) A stainless steel screen is placed over the slots, covering the slots and preventing the eggs from washing out the slots while still allowing for overflow.  The tank's influent delivery line discharges directly into the incubator.  A disposable pipette bubbles air to gently agitate the embryos.   (2)  Threaded Cap Incubator.

      Figure 2.  Threaded Cap Incubator.  This incubator consists of a transected glass centrifuge tube, connected by a stainless steel sleeve and held in place by the centrifuge screw top cap.  A small glass or stainless steel tube projects through the cap and is positioned near the rounded bottom, gently bubbling air to suspend the eggs and reducing between-egg transmission of saprophytic fungal infections while also facilitating chemical exchange between the incubator and the holding tank.

(3)  Syringe Style Incubator.

Figure 3.  Syringe Style Incubator.  This incubator consists of a fluid dispensing syringe assembly containing stainless steel screens located at the top and bottom of the syringe, ensuring that the eggs are contained within the syringe body. A series of valves allow the flow to the incubator to be precisely regulated and/or diverted to the aquaria or to drain as needed. The flow of fluid through the incubator should be sufficient to provide agitation to the eggs.

(c)   Isolation Chambers (Figure 4).  From Study Week 6 through Study Week 11 of the F1 generation, twelve fish are held in each replicate tank.  The genotypic sex of each fish is determined during Study Week 12.    Each fish will have a small sample of the caudal fin taken to determine its sex.  The DMY analysis has a turnaround time of 24-48 hours.  The fish need to be held as identifiable individuals until their genetic sex has been determined and they can be assigned to a breeding pair or sacrificed for the subadult endpoints.  Isolation chambers are used to hold the fish, with two fish contained in each chamber.  The caudal fin of each fish is differentially cut with one a dorsal and the other receiving a ventral cut.  Six chambers are set up in each aquarium.  The placement of the chamber in the replicate aquarium is pre-determined to maintain the identity of each fish.

Figure 4. Isolation Chamber Illustration. On the left, a single chamber.  On the right, aquarium with six chambers.

(d)  Temperature Control and Monitoring.  Fecundity and growth endpoints require precise temperature control and monitoring.  A dry system using low wattage rubber silicon heaters controlled by a digital temperature controller is recommended (Figure 5).  The heating system should be sized to avoid large temperature swings if a component fails.  To further aid in temperature control, the aquaria should receive treatment water that is temperature controlled at the nominal test temperature.  The tank heater system only inputs heat to compensate for ambient loss.  It is also recommended that the heating system is linked to a data collection system that provides real time temperature monitoring and data logging for each aquaria.

Figure 5.  Tank temperature and monitoring display.

(e)  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.

  (1)  Multi-channel Peristaltic Pump (Figure 6).

Figure 6. Multi-channel Peristaltic Pump.  One system that meets these requirements is a multi-channel peristaltic pump system which can deliver treatment water from the treatment reservoir to each replicate exposure aquarium.  These systems can reliably deliver flows up to 50 ml/min/channel.  Delivery lines should be sterilized on a set maintenance schedule to control 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 treatment flow is controlled by the sped of the pump which continuously prepares treatment solution.  Treatment solution is sequentially dispensed to the replicate tanks by the opening of a solenoid directing flow to a tank.         (f)  Chemical Dilution System.  The precise preparation of toxicant solutions to be delivered to exposure tanks is critical to the success of a long-term bioassay.  The design considerations for a toxicant preparation system are:

 * The preparation of treatments should be independent.  This enhances study design flexibility by allowing the adjustment of individual treatment concentrations, the addition or elimination of treatments, and the selection of concentration gradients based on the experimental design and not on system limitations.  * The ability to produce a large range of treatment concentrations.  * The toxicant should have short residence time in the system.  The toxicant should undergo continuous dilution with rapid delivery to the exposure tanks.    * Excessive chemical should not be sent to waste.  * If the system fails the toxicant levels should drop.

One toxicant preparation system meeting these requirements incorporates dual headed positive displacement pumps which have independent pump heads linked by a common drive motor.  Each treatment solution is prepared by a dedicated pump (Figure 8).  The pump links a flow of dilution water to a flow of toxicant which are combined at a T fitting and directed through a static mixer into the treatment reservoir.  The ratio of dilution water to toxicant is set by adjusting the piston displacement volume of the pump heads.  The piston adjustment is independent for each pump head and flow ratios of 1:1 to 200:1 can be set up.  This range can be expanded by swapping in higher or lower flow pump heads.  The flow to the treatment reservoir is controlled by the speed of the drive motor.  The pump speed can be manually adjusted or controlled with a circuit.  Treatment solutions have been diluted at rates from 10 to 260 ml / min.

Figure 8.  Example of toxicant preparation system.           APPENDIX 3:  Example Daily Calendar.

Medaka Extend One-Generation Reproduction Test  Daily Protocol Enter Fertilization Date (F0 Generation): 4/9/2012   Chemical:

    Enter Start Date (move F0 breeding pairs into system): 6/25/2012   Temp: 25o C           Event  (1 row for each day of study) Day Date Calendar Day Test Wk Test DayTest 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

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

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         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

APPENDIX 4:  Example Protocol for DNA Extraction from Fin Clip Tissues

(a)  Background.  In the MEOGRT, subadult medaka are assessed for the presence or absence of the dmy gene in order to 1) setup up breeding pairs of one XX and one XY fish, and 2) provide DMY data for each of the fish sacrificed at this time.  The first step in this process is to extract DNA from a tissue sample, typically a fin clip, of each fish. This can be done using commercially available kits that are based upon selective binding of DNA by a silica-gel-based membrane or other suitable methods of DNA extraction.

The example protocol presented is for DNA extraction from fin clips based on procedures used at the U.S. Environmental Protection Agency laboratory in Duluth, MN.  Specific products and/or equipment listed can be substituted with comparable materials.

(b)  Major Materials and Reagents.  DNA may be extracted by a variety of methods.  In this example, a commercial DNA extract kit, DNeasy Blood and Tissue Kit (Qiagen), is used to extract DNA from a fin clip tissue sample.

   * DNA extraction kit ( (DNeasy Blood and Tissue Kit, Qiagen)    * Microcentrifuge and tubes

(c)  Protocol

(1)  Tissue Collection.  With the fish under anesthesia, remove a small piece (~1 mm square) of tail fin, place in a 1.5 ml microcentrifuge tube and immediately quench freeze in liquid nitrogen.  Fins are stored at less than -20° C until further use.  If tissue is going to be immediately processed, freezing can be omitted and the tissue can be processed as indicated below.

(2)  DNA Extraction.  Extract DNA from fin clip samples using the DNeasy Blood and Tissue Kit.  Add 180 ul Buffer ATL to fin clip followed by 20 μl proteinase K, and vortex.  Alternatively, Buffer ATL and proteinase K can be premixed immediately before sampling large numbers of fin clips and 200 μl added to fin clip.  Incubate microcentrifuge tubes at 56°C until tissue is lysed (usually takes approximately 45 minutes but can be left overnight), vortex for 15 seconds, and add 200 μl Buffer AL before vortexing another time.  Add 200 μl of 100% ethanol, and then vortex briefly.  Pipet resulting mixture onto a DNeasy Mini spin column, centrifuge at greater than 6000 x g for 1 minute, and discard the flow-through/collection tube.   Wash bound DNA with 500 μl Buffer AW1, centrifuge at greater than 6000 x g for 1 minute (discard 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 tube, pipet 100 μl Buffer AE onto the column, incubate at room temperature for 1 minute, and centrifuge at 6000 x g for 1 minute to elute DNA.  Store DNA at less than -20°C until used.     	 Quantification of the extracted DNA is unnecessary because it will be used in a qualitative test to detect the presence or absence of the dmy gene (see Appendix 5). 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.

APPENDIX 5:   Example Real-Time PCR Protocol to Detect dmy Gene in Japanese medaka

      (a)  Background.  In the MEOGRT, subadult medaka are assessed for the presence or absence of the dmy gene (GenBank: AB071534.1) in order to 1) setup up breeding pairs of one XX and one XY fish, and 2) provide DMY data for each of the fish sacrificed at this time.  After the DNA is extracted (see Appendix 3, the presence (or absence) of dmy, the medaka male determining gene, is assessed in each sampleThe dmy gene is present on the Y chromosome of every male somatic cell, but is not present in any of the female cells providing for a definitive test for genotypic sex.  A DNA sample that is positive for the dmy gene is from a genotypic male while a DNA sample that is negative for the dmy  gene is from a genotypic female (Matsuda et al., 2002).  These results are independent of phenotypic sex which may have been altered by exposure to a test chemical that might disrupt endocrine hormone pathways. The protocol discussed below is an example of real-time PCR assay to detect the dmy gene.  The example procedure is based on procedures used at the U.S. Environmental Protection Agency laboratory in Duluth, MN.  Specific products and/or equipment listed can be substituted with comparable materials.  In this example, a Taqman(R) assay is used to detect amplification of the dmy gene.