DRAFT: Medaka Reproduction Test

Guideline March 27, 2013

Medaka Reproduction

Test Guideline

1 April 2013
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Medaka Reproduction Test Guideline

OBJECTIVE

This test guideline describes a fish multigeneration test that can be used to evaluate the 

potential chronic effects of chemicals on fish populations. The method gives primary 

emphasis to potential population relevant effects (namely, adverse impacts on survival, 

development, growth and reproduction) for the calculation of a No-Observed Effect 

Concentration (NOEC). These effect observations are augmented by evaluating secondary 

mechanistic biomarker responses (namely, vitellogenin and gonad histology). The method is 

applicable to a variety of chemicals, including endocrine disrupters and general toxicants. 

The medaka (Oryzias latipes) is the appropriate species for use in this test guideline.

GLOSSARY OF TERMS

wpf= weeks post fertilization; dph = days post-hatch; SSC = secondary sex characteristics; 

VTG = vitellogenin 

INTRODUCTION

The Medaka Reproduction Test (MRT) guideline describes a definitive test based on fish 

exposed over multiple generations to give data relevant to ecological risk assessment of 

endocrine disrupting chemicals (EDCs). A fish full life-cycle test for ecotoxicological studies 

has already been established by the U.S. Environmental Protection Agency (U.S. EPA) in 

fathead minnows (Pimephales promelas) and sheepshead minnows (Cyprinodon variegatus)

(U.S. EPA, 1986). The present protocol in this guideline is derived from work on medaka 

(Oryzias latipes) by the U.S. EPA with supporting work by the Chemicals Evaluation and 

Research Institute, Japan (CERI) and National Institute for Environmental Studies, Japan

(NIES). 

This test guideline measures several biological endpoints. Primary emphasis is given to 

potential adverse effects on population relevant parameters including survival, gross 

development, growth and reproduction. Secondarily, in order to provide secondary

mechanistic information and provide linkage between results from other kinds of field and 

laboratory studies, where there is a priori evidence for a chemical having potential endocrine 

disruptor activity (e.g. androgenic or oestrogenic activity) then other useful information can be 

obtained by measuring vitellogenin (VTG) levels, phenotypic secondary sex characteristics

[SSC], genetic sex, and evaluating histopathology.

PRINCIPAL OF THE TEST

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

development in medaka exposed throughout multiple generations of their life-cycle. The data 

can be used to support Tier 2 of the Endocrine Disruptor Screening Program in response to the 

Food Quality Protection Act. The test method begins by exposing adult fish (the P or F0 
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generation) to the test chemical and continues through reproductive development and 

reproduction in the F1 generation followed by an assessment of hatch success in the F2 

generation; thus the assay allows evaluation of both structural and activational endocrine 

pathways.

 This test, which primarily measuresthe reproductive performance of medaka is started by 

exposing young reproductively active fish for 3 weeks, during which the chemical is 

distributed to the gametes and tissues of the parental generation (F0). At the beginning of the 

fourth week, eggs are collected to start the F1 generation. Fish in the F1 generation are reared 

for a total of 15 weeks post fertilization (wpf). Fish from the F1 generation are sampled at 9 

wpf for evaluation of developmental endpoints. F1 reproduction is assessed for 21 continuous 

days during the period of 12-14 wpf. 

DESCRIPTION OF THE METHOD

Test animals

The test species is medaka (Oryzias latipes). The medaka is readily induced to breed in 

captivity and published methods exist for its culture, as well as short-term lethality, early 

life-stage and full life-cycle tests. All fish will be maintained on a 16-hr light:8- hr dark

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

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

live brine shrimp (Artemia) nauplii throughout the test.

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

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

actively spawning. For the whole group of fish used in the test, 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. The age of the fish selected should be 

within the range of 11 to 15 weeks post fertilization (females > 300 mg; males > 250 mg).

 Test fish 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 will interfere with reliable spawning. Fish should be fed brine shrimp twice per 

day throughout the holding period and during the exposure phase.

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

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

to the test system: reject the entire batch;

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

days; if more than 5% mortality during second seven days, reject the entire batch;

• Mortalities of less than 5% of population in seven days: accept the batch.
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 Fish should not receive treatment for disease in the two-week acclimation period preceding 

the test, during pre-exposure, or during the exposure period. Moribund fish or fish with 

clinical signs of disease should not be used.

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 substance) 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. Some chemical characteristics of 

acceptable dilution water are listed in Annex 1.

Exposure system

 The design of the exposure system is optional. Glass or other chemically inert material 

should be used for construction of the test system. The literature describes several different 

exposure systems used in medaka testing (Yokota et al., 2000, 2001a; Kang et al., 2002; Seki 

et al., 2002). The chemical dilution system may use positive displacement pumps to measure 

and mix the appropriate ratio of stock chemical solution with dilution water into a dilution cell 

(aquarium). These dilutions may be delivered to the various exposure aquariums using 

multichannel peristaltic pumps. The flow rate of the stock solution should be calibrated in 

accordance with analytical confirmation of the test solutions before the initiation of exposure, 

and checked volumetrically periodically during the test. The test solution in each chamber 

shall be renewed adequately based on the requirements of the fish and on the stability of the 

test chemical.

Experimental Design

 It is recommended to use a minimum of five chemical concentrations plus control(s). Prior 

knowledge of the test substance (e.g. results of fish toxicity tests and a range-finding test) 

should help in selection of appropriate test concentrations. 

 If necessary, a range-finding test may be conducted under conditions (water quality, test 

system, animal loading) similar to those used for the definitive test. For the tests with a strong 

estrogen or androgen, histological measurements are much more responsive than subchronic

effects, therefore, the highest concentration used in the definitive test should be selected to 

cause considerable sex reversal. The lowest concentration should be a factor of 10- to 

100-times lower than the highest concentration. The use of five concentrations in this test 

enables the evaluation of dose-response relationships, and it also provides data for 

determining the lowest-observed-effect concentration and no-observed-effect concentration,

which are necessary for risk assessment. A minimum of six replicate test chambers in each 

treatment concentration and a minimum of twelve replicate test chambers in the control 

treatment, is recommended during the growth and developmental portions of the test. During 
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the assessment of reproduction, in which each test chamber houses one reproductive pair, the 

number of test chambers in the both the treatment concentrations and controls should be 

doubled to ensure an adequate statistical evaluation of possible treatment related effects on 

fecundity and fertility. It has been demonstrated that this replication structure enables the 

detection of statistically-significant differences for the majority of the endpoints in control fish 

versus animals treated with “model” EDCs, including 4-nonylphenol (Yokota et al., 2001a), 

4-tert-pentylphenol (Seki et al., 2002 [submitted]), ethinylestradiol, methyltestosterone, 

4-tert-octylphenol and 17β-estradiol.

Administration of test substance via water and analytical determination

The recommended route of exposure for the test is via chemical transfer from the water 

column across the gill epithelium into the fish. This is accomplished by exposing the fish to 

the test substance dissolved in the water being delivered to the test aquarium.

 Test solutions of the chosen concentrations are prepared by dilution of a stock solution. The 

stock solution can be prepared by simply mixing or agitating the test substance in the dilution 

water by using mechanical means (e.g. stirring or ultrasonication). Saturation columns can 

also used for achieving a suitable concentrated stock solution. It is recommended that, 

whenever possible, solvents not be used to generate stock solutions for conducting chemical 

exposures via the water. 

 In some cases it may be necessary to use solvents to generate stock solutions for aqueous 

testing; this could occur when a chemical is very insoluble, or so expensive/limited in 

availability that the use of saturators is not practical. Generally, examples of suitable solvents 

in fish chronic toxicity test are acetone, dimethylformamide, dimethylsulfoxide, methanol and 

triethylene glycol. However, there are relatively few studies on the possible effects of these 

solvents on the fish endocrine system. Hence, it is essential that any test utilizing a carrier 

solvent include both solvent-exposed and non-exposed controls.

Analytical determination and measurements

 During the test the chemical concentrations in each of the treatments should determined 

every other week at a minimum, and the flow to each test aquariums should be verified daily 

and measured at least weekly. Preceding the initiation of the test exposure it is important to 

verify that the chemical dilution and delivery system can provide the desired chemical 

concentrations to the exposure aquariums. The concentrations of the test chemical will likely 

need to be measured in stock bottles (or saturator column eluates), 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 chemical.

 After the exposure is initiated, it is important to monitor the concentration of the test 

chemical in the exposure aquariums frequently enough to allow accurate characterization of 

the actual concentrations the test animals were exposed to, 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 chemical concentrations in each treatment as 

necessary. Weekly sampling of at least two replicate aquariums from each treatment is usually 
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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 systematically rotated from one the other possible replicates. This sampling strategy 

will provide estimates of chemical concentration in each treatment through time as well as 

estimates of the variation of the within-treatment concentrations. It is also useful to evaluate 

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

measurement replication and chemical standards. For chemicals 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. Asuggested guideline for analytical sampling is 

presented in Annex 2. 

PERFORMANCE OF THE TEST

 Briefly, the test consists of continuously exposing multiple generations of fish to the test 

chemical starting with young adults. To provide data for determination of lowest- and 

no-effect concentrations for a series of endpoints, there are 6 treatments; five chemical 

concentration groups and a control group. The number of replicates within each treatment 

varies throughout the test. Initially, there are six replicates for each concentration during the 

period of the test from embryo through development to subadults (9 wpf) and 12 control 

replicates for this same period. Replication is then doubled for the assessment of reproduction. 

The increased replication for evaluating reproduction is necessary to provide robust statistical 

evaluation of treatment effects on that important endpoint. The measured endpoints include 

fecundity and fertility for evaluation of possible population-level effects, liver vitellogenin 

mRNA levels, the number of papillae on the anal fin (a marker of sexual phenotype), 

histological evaluation of gonadal sex and intersex, and finally, a histopathology evaluation of 

kidney, liver, gonad, and other tissues (see endpoint list in Table 2). All of these endpoints 

are evaluated in the context of the genetic sex of the individual. Non-endocrine-mediated 

toxicity endpoints such as embryo hatch, growth, and survival are also measured.Throughout 

the bioassay, chemical concentrations are held constant to allow endpoint sensitivity 

comparisons across and within generations. Thus, although not discussed here in detail, 

precise dependable flow-through exposure and dilution systems as well as high-quality 

analytical chemistry is an integral part of protocol implementation.

General Experimental Design

 The age of all fish in the MMT test is specified in weeks post-fertilization (wpf). The 

temperature throughout the test is 25.5° C, and the photoperiod is 16h light and 8h dark. The 

light intensity should be between 100-500 lux during the light period, but may be increased up 

to 1,000 lux during test maintenance and sample collection. The control and exposure water 

flow to each 2 L aquarium is 20 ml/min. The general design employs 6 treatments including 

controls hereafter designated as T1 (controls with clean dilution water) through T6 (the 

highest treatment concentration). Generally, the nominal concentration ratio of the test 

chemical between adjacent treatment levels is 0.5. A complete summary of test parameters 

can be found in Table 1. Adherence to these specifications should result in control fish with 

endpoint values similar to those listed in Annex 3.
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Table 1. Test Conditions for the Proposed Medaka Reproduction Test (MRT)

Recommended species Medaka (Oryzias latipes)

Test type Continuous flow-through

Water temperature 25.5 ± 0.5 oC 

Illumination quality Fluorescent bulbs (wide spectrum and ~150 lumens)

Photoperiod 16 h light, 8 h dark

Loading rate F0- 2 fish/replicate; F1 and F2- initiated with maximum of 20 larvae/replicate eventually culled to 

2 fish/replicate

Test replicate chamber size 18x9x15 cm 

Test replicate solution volume ~1.8 liters 

Volume exchanges of test solutions 16 volume replacements/day (20 mls/min flow).

Age of test organisms at initiation F0-11 weeks post-fertilization, F1 and F2 continuously from fertilization

No. of organisms per replicate F0- 2 adults; F1 and F2- initiated with maximum of 20 larval fish eventually culled to 2 adults

No. of treatments 6 (5 toxicant treatment plus 1 control)

No. replicates per treatment 6 per treatment for each toxicant treatment; 12 for control treatment; replication structure 

doubled for fecundity assessment

No. of organisms per test concentration F0- 24 for each test concentration and 48 for controls; F1 and F2- start with a maximum of 120 

and eventually cull to 48 for fecundity assessment.

Feeding regime Fish were fed 24-hour old brine shrimp (Artemia spp.) nauplii at the following rates:

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 1.0 Day 16-19 5.6

Day 6 1.3 Week 4 7.7

Day 7 1.7 Week 5 9.0

Day 8 2.2 Week 6 11.0

Day 9 2.8 Week 7 13.5

Day 10 3.5 Week 8-15 22.5

Day 11 4.2

Aeration None unless DO reaches <60% saturation

Dilution water 50 micron sand-filtered Lake Superior Water, UV sterilized in the lab and further filtered to 5 

microns and degassed to 8.0 mg O2/L.

Exposure period Trans-generational test: F0 from 11-15 wpf, F1 and F2 from 0-15 wpf

Primary endpoints Survival: F1 from 1-3 wpf and from 3-15 wpf

Growth : length, weight, (F1 at 9 wpf and 15 wpf 

Hatch: F1and F2 only

Anal Fin Papillae: F1 assessed at 9 wpf and 15 wpf 

Fecundity: F1 assessed daily for 21 days, 12-14 wpf

Fertility: F1 assessed daily for 21 days, 12-14 wpf

Vitellogenin: F1 only at 9wpf
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Optional endpoints Time to first spawn for F1 generation.

Test acceptability Dissolved oxygen >60% of saturation; mean temperature of 25.5 ± 0.5 o

C; ≥70% survival in 

control treatments of each generation; successful reproduction in at least 65% of control females 

with mean daily fecundity in the control treatment of at least 15 eggs.

Start of Test

 The test is started by exposing young adult fish (F0) to the test chemical. The F0 fish are 

selected based on their age, from 11 to 15 wpf old, weight, at least 300 mg for females and 

250 mg for males, and having sexual genotype and phenotypes that match. While no 

specific culturing protocol is recommended, appropriate husbandry practices should be 

followed. At the USEPA laboratory in Duluth, MN, medaka are reared in 2L aquariums with 

120 larval fish per aquarium until 4 wpf, and 10 fish per aquarium until 9 wpf. At this time 

breeding pairs are selected, based on sexual phenotype, and placed in 2L aquariums (one pair 

per aquarium). These breeding pairs can be used as F0 fish for the start of the MMT. However, 

before the start of the test, the genetic sex of the breeding pairs should be determined. 

Breeding pairs consisting of an XX and an XY fish and matching sex phenotypes should be 

used to start the test. Be sure that there are no XX males present in your breeding pairs. Lastly, 

just before the test is started it is helpful to briefly assessthe breeding pairs for spawning 

competency. Only competent female-male pairs that meet the above specifications are used to 

start the MMT. The MMT begins by randomly placing the selected pairs into the replicate 

treatment aquariums (T1-T6) and starting the appropriate flow of chemical to each aquarium.

Feeding Protocol

Relatively large amounts of high quality food are fed throughout the MMT to optimize fish 

growth. Rapid growth is necessary to adequately assess reproductive development and 

reproduction in the relatively short MMT. Newly hatched brine shrimp (Artemia) are a 

recommended food that can be fed on a dry weight (shrimp) to wet weight (fish) ratio as 

suggested in supplemental tables. Test fish are fed brine shrimp exclusively on a 

weight-weight basis optimized for growth. In the event of mortality, the food delivered to 

each aquarium is reduced in proportion to the number of fish in the aquarium.

Egg Collection for Starting F1 and F2 Generations

 Eggs are collected during the first two days of study weeks 4 and 18 for initiating the F1 

and F2 generations respectively. After spawning, all eggs are collected by either carefully 

removing attached eggs from the female or by siphoning the eggs from the bottom of each 

aquaria. The eggs from each treatment are are pooled and then fertile, healthy eggs are 

randomly selected for incubation. A maximum of 120 eggs from each treatment 

concentration and 240 from the controls are placed in an appropriate incubating device, (see 

Figure 1 for an example incubator used by MED-Duluth) until hatch. It is preferable that the 

eggs are collected on a single day to initiate the F1 generation however, if exposure to the test 

chemical causes reduced fecundity, the eggs may be collected over two days. The incubators 

must continue to receive an adequate flow of the test chemical concentration for the duration 

of the incubation period. These incubators can be located either in separate “incubator” 

aquaria or in the same aquaria containing the breeding pairs. If the incubators are placed in the 
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same aquaria that contain the breeding pairs and if the fish are fed, care must be taken to 

ensure that the incubators do not become fouled with brine shrimp. 

Figure 1. Example egg incubator used to hatch small numbers (maximum of 20) medaka eggs within 

the exposure system. The incubator consists of a transected glass centrifuge tube, connected by a 

stainless steel mesh sleeve and held in place by the centrifuge screw top cap. A small glass or 

stainless tube projects through the cap and is positioned near the rounded incubator bottom. Gentle 

bubbling with air provides agitation of the eggs which helps reduce between-egg transmission of 

saprophytic fungal infections and also facilitates chemical exchange between the incubator and the 

holding aquarium. 

Hatching Eggs

 Eggs are continually agitated within the incubator by constant aeration. Incubators are 

checked daily for mortalities. Mortalities are recorded and all dead eggs are removed from 

the incubators. On the 8th day post fertilization, the aeration is stopped, causing the eggs to 

settle to the bottom of the incubator. This promotes hatching, typically over the next two 

days. For each treatment, hatchlings are counted, pooled, and systematically distributed to 

each of the replicate aquaria . Embryos that have not hatched by the 14th day post fertilization 

are considered non-viable and discarded.

Fin Clipping and Determination of Genotypic Sex to Setup Breeding Pairs

 Determination of genotypic sex via fin clips in the F1 generation is done in Study Week 12. 
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Fish are anesthetized and either the dorsal or ventral tip of the caudal fin of each fish is 

clipped. Fish are then segregated in a chamber, two fish per chamber, each with a different fin 

clip pattern. The chamber is then placed in the original aquarium. When complete, each 

aquarium contains 6 segregated fish pairs, with each member of a segregated pair having a 

different fin clip. This allows each fish within an aquarium to be individual identified and 

matched with a particular fin clip.

 The genotypic sex of medaka is determined by an identified and sequenced gene (DMY) 

which is located on the Y chromosome. DNA from each fin clip is extracted and the 

presence or absence of DMY is determined by PCR methods. The presence of DMY 

indicates a XY individual, regardless of phenotype,

while the absence of DMY indicates a XX individual, regardless of phenotype.

 If two of the six of the segregated pairs are XX-XY, then they are selected as the breeding 

pair for the reproductive assessment and placed in separate aquaria or in a single divided 

aquarium. If more than two of the segregated pairs are XX-XY, two of them are randomly 

selected as breeding pairs. If there are not enough XX-XY pairs present, then XX fish and 

XY fish are randomly selected from the other segregated pairs within that aquarium to set up 

the required number of breeding pairs. If there are not enough XX and XY individuals 

present within an aquarium, then the needed genotype(s) is randomly selected from another 

replicate.Any fish that is obviously in poor health should not be selected for a breeding pair.

Sampling of F1 Non-breeding Pair Fish 

 The F1 pairs not selected as breeding pairs are sacrificed for various endpoints (see Table 2, 

Sub-adults) in Study Week 12. It is extremely important that the fish are handled in such a

way so that the genotypic sex of each fish is determined and can be traced back to that fish. 

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

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

growth, liver vitellogenin mRNA, anal fin papillae number, kidney pathology and 

histologically-determined gonad phenotype (i.e., testis, ovary, or gonadal intersex). The liver 

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

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

appropriate fixative (ex. Davidson’s) so that anal fin papillae may be counted at a later date. 

The remaining tissue is fixed in Bouin’s, embedded in paraffin, and sectioned to histologically 

determine the gonad phenotype. It is recommended that at this sampling another tissue sample 

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

earlier DMY analysis in the event that genotype and phenotypic endpoints are nonconcordant.

Vitellogenin (VTG)

 Vitellogenin (Vtg), a common biomarker of EDC exposure, is measured by determining 

the Vtg mRNA copy number per ng of total RNA in the liver by quantitative PCR (QPCR). 

QPCR is highly sensitive and has a large dynamic range (~7 orders of magnitude), making it 

an ideal tool for efficiently assessing concentrations of a target gene. Specifically, Vtg is 

measured in adults at the end of each generation (F0, F1, and F2; and in subadults in F1 and 

F2. The per treatment data is categorized by gender genotype (XX or XY) and then 
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compared to the gender-specific control data from each particular generation and life-stage. 

Fecundity and Fertility Assessment

 Fecundity and fertility are assessed in Study Weeks 15 through 17 for the F1 generation . 

Eggs are collected daily from each breeding pair for 21 days. Egg clumps are gently removed 

from netted females or siphoned from the bottom of the aquarium each morning. The 

number of eggs spawned and the number of fertile eggs are determined for each pair. 

Sampling of Breeding Pairs: Adults

 At Study Week 18 (15 wpf) the F1 adults are sacrificed and various endpoints (Table 2, 

Adults) are assessed. While the tail is removed and fixed to assess the number of anal fin 

papillae, no other tissue dissection is performed. The body cavity is opened to allow 

perfusion with Bouin’s fixative prior to submersing the entire body in the fixative. Each fish 

is evaluated histologically for pathology in the gonads, kidneys and liver tissue. The gonad 

phenotype is also derived from this evaluation. 

Endpoint Overview

 The MRT test provides data that can be used to simultaneously evaluate two general types 

of adverse outcome pathways (AOPs) ending in reproductive impairment: a) 

endocrine-mediated pathways involving disruption of the hypothalamus-pituitary-gonadal 

(HPG) endocrine axis; and, b) pathways that cause reductions in survival and growth through 

non-endocrine mediated toxicity. Test data provides information for evaluating adverse 

outcomes from either endocrine-mediated or non-endocrine mediated effects, or both. The 

MRT endpoint data are listed in Table 2. Some of the EDC endpoints, such as the presence of 

anal fin papillae in medaka males, are effect biomarkers only minimally linked to adverse 

reproductive outcomes; whereas other EDC endpoints such as fecundity and fertility can be 

directly linked to adverse outcomes through life-history translational models and, with 

possible additional links, to population models. Endpoints typically measured in chronic 

toxicity tests such as the full life-cycle test, and the early life-stage (ELS) test are also included 

in the MMT and can be used to evaluate the hazards posed by both non-endocrine mediated 

toxic modes of action and endocrine-mediated toxicity pathways.

Table 2. Endpoint overview of the Medaka Reproduction Test.

Life-stage Endpoint EndocrinemediatedNon-EndocrineMediatedDirect Population 

Relevance

ELS (1 wpf) Hatch Y Y

ELS (4 wpf) Survival Y Y

Sub-adult (9 wpf) Survival Y Y

Growth 

(weight/length)

Y

Gonad phenotype Y Y

Vitellogenin (Vtg) Y
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Anal fin papillae Y

Histopathology

Gonad

Kidney

Y Y

Y

Adult (12-14 wpf) Fecundity Y Y Y

Fertility Y Y Y

Adult (15 wpf) Survival Y Y

Growth 

(weight/length)

Y

Gonad phenotype Y Y

Anal fin papillae Y

Histopathology

 gonad

 liver

 kidney

 other

Y Y

Y

Y

Y

Medaka Reproduction Test Timeline

 A timeline for the MRT is illustrated in Figure 2. The test includes one complete generation

of medaka lasting for 15 weeks and partial adult and embryonic components of two other 

generations. The activity through the course of the test is summarized here. The methods 

section contains the necessary details to accomplish each of the test activities. In addition, an 

Excel spreadsheet is available as a separate file that provides detailed, day-by-day instructions 

for the entire test. 

Study Weeks 1-3: The first generation (F0) young adult spawning fish (11 to 15 week post 

fertilization; females > 300 mg; males > 250 mg) are exposed for three weeks during which 

time the developing oocytes and sperm are “loaded” with the test agent and associated 

maternal/paternal factors as defined by the relative treatment levels. On Study Day 1, a 

minimum of six female-male breeding pairs are placed in an exposure system that has six 

treatment levels including controls with six replicate aquaria per treatment concentration and 

12 replicate control aquaria. Each replicate aquaria houses a single breeding pair of fish for a 

test total of 42 breeding pairs 

Study Week 4: On Study Day 21, eggs are removed from both the aquaria and the females 

to guarantee an accurate collection the next day. The following day (Study Day 22), eggs 

are collected and pooled from each treatment and systematically distributed to a suitable 

incubation vessel(s) with no more than 120 eggs for each treatment concentration and 240

eggs for the controls allowed to incubate. The eggs may be incubated in separate “incubator 

aquaria” set up for each treatment or in the same replicate aquaria that will contain the hatched, 

larval fish. Ideally, all eggs used to initiate the F1 generation should be collected on the 

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

viable eggs is collected on the first day.

Study Weeks 5-6: On Study Day 24, the F0 breeding pairs are sacrificed. On Study Day 

30, the aeration

(agitation) of the incubating eggs is turned off to expedite hatching. As eggs hatch on each 

day, hatchlings from each treatment are pooled and then systematically distributed to each 

replicate aquarium within a specific treatment with no more than 12 hatchlings placed into 
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each aquarium. The appropriate data is collected to calculate the hatching success of each 

incubator. On Study Day 36, any unhatched eggs from the first day of egg collection are 

discarded. If eggs were collected over a two day period, unhatched eggs from the second day 

of collection are discarded on Study Day 37.

Study Weeks 7-11: On Study Day 44 survival of the larval fish to this point is recorded. 

The exposure continues as the larval fish develop into sub-adults. 

Study Week 12: On Study Day 78, a small sample is taken from the caudal fin of each fish 

to determine the genotypic sex of the individual. This information is used to establish 

female-male breeding pairs. On Study Days 80 and 81 after the genotypic sex of each fish is 

determined, two breeding pairs per replicate are randomly established while the 8 (nominal) 

remaining fish per replicate are sampled for the various endpoints outlined in Table 2, 

sub-adults. 

Study Weeks 13-14: The exposure continues as the sub-adult breeding pairs develop into 

adults. 

Study Weeks 15-17: Spawned eggs are collected daily for 21 consecutive days, Study Days 

99-119, and counted and assessed for fertility. On Study Day 119, eggs are removed from 

both the aquaria and the females to guarantee an accurate collection the next day. 

Study Week 18: On study day 120 eggs are collected and pooled from each treatment and 

systematically distributed to a suitable incubation vessel(s) with no more than 120 eggs for 

each treatment concentration and 240 eggs for the controls allowed to incubate. The eggs may 

be incubated in separate “incubator aquaria” set up for each treatment or in the same replicate 

aquaria that will contain the hatched, larval fish. Ideally, all eggs used to determine F2 hatch 

success should be collected on the same day however, a second day of collection may be 

necessary if an insufficient number of viable eggs is collected on the first day. 

After one or two days of egg collection, the F1 breeding pairs are terminated and analyzed for 

the endpoints in Table 2, adults. 

Study Week 19: On Study Day 128, the aeration (agitation) of the incubating eggs is turned 

off to expedite hatching. The number of hatchlings are recorded to determine F2 hatch 

success.
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Exposure and Endpoint Timeline

Gen

F0 1 2 3 4

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

F2 1 2

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

Embryo Larvae Juvenile Subadult Adult

Endpoints

Fecundity F1 Experimental design: 7 blocks where 

1 block=12 replicate aquaria for 

fecundity and fertility 

measurements and 6 replicate 

aquaria for the embryo thru 

subadult endpoints

5 blocks for chemical treatments

2 blocks for control treatments

Fertility F1

Hatch F1 F2

Survival F1 F1 F1

Growth 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

Figure 2. Overview of the Medaka Reproduction Test timeline and endpoint summary.

VALIDITY OF THE TEST

 This test has not been performed extensively, but based on the limited experience with 

medaka and this test protocol to date, the following criteria for the test acceptability are 

suggested:

• The dissolved oxygen concentration must be between 60% and 100% of the air 

saturation value throughout the test;

• The recommended temperature and variance throughout the test is 25.5°C ± 

0.5°C. Brief excursion from the mean by individual aquaria must not be more than 

2°C. Replicates within a treatment must not be statistically different from each other, 

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

•

• There should be at least 65% survival of control animals after hatching through 21-d 

posthatch (3 wpf) and greater than 85% survival from 3 wpf to 8 wpf in the F1

generation.

As experience is gained with this test, additional biological performance criteria can be 

incorporated. 

DATA REPORTING
14

Statistical analysis

 Statistical analyses of the data should preferably follow procedures described in the 

document 

Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application 

(OECD 2006). Data for continuous endpoints should be assessed for normality (preferably 

using the Shapiro-Wilk or Anderson-Darling test) and variance homogeneity (preferably using 

the Levene test). Both tests are performed on the residuals from an ANOVA. Expert 

judgment can be used in lieu of these formal tests for normality and variance homogeneity, 

though formal tests are preferred. Where non-normality or variance heterogeneity is found, 

a normalizing, variance stabilizing transformation should be sought. If the data (perhaps 

after a transformation) are normally distributed with homogeneous variance, a significant 

treatment effect is determined from Dunnett’s test. If the data (perhaps after a 

transformation) are normally distributed with heterogeneous variance, a significant treatment 

effect is determined from the Tamhane-Dunnett or T3 test or from the 

Mann-Whitney-Wilcoxon U test. Where no normalizing transformation can be found, a 

significant treatment effect is determined from the Mann-Whitney-Wilcoxon U test using a 

Bonferroni-Holm adjustment to the p-values. The Dunnett test is applied independently of

any ANOVA F-test and the Mann-Whitney test is applied independently of any overall 

Kruskall-Wallis test.

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 >5 mortalities in any replicate that cannot be explained by technical error. 

Other signs of overt toxicity include hemorrhage, abnormal behaviors, abnormal swimming 

patterns, anorexia, and any other clinical signs of disease. For sub-lethal signs of toxicity, 

qualitative evaluations may be necessary, and should always be made in reference to the clean 

water control group. 

Solvent controls 

 The use of a solvent should only be considered as a last resort, when all other chemical 

delivery options have been considered. If a solvent is used, then a clean water control must 

be run in concert. At the termination of the test, an evaluation of the potential effects of the 

solvent must be performed. This is done through a statistical comparison of the solvent 

control group and the clean water control group. The most relevant endpoints for 

consideration in this analysis are growth determinants (weight and length), as these can be 

affected through generalized toxicities. If statistically significant differences are detected in 

these endpoints between the clean water control and solvent control groups, determine the 

study endpoints for the response measures using the clean water control. If there is no 

statistically significant difference between the clean water control and solvent control for all 

measured response variables, determine the study endpoints for the response measures using 

the pooled dilution-water and solvent controls. Any endpoints that are significantly affected

by the test chemical should be reported. 
15

Test report

 The test report must include the following:

Test substance:

-physical nature and, where relevant, physicochemical properties;

chemical identification data.

Test species:

-scientific name, strain, source and method of collection of the fertilized eggs and subsequent 

handling. 

Test conditions:

-photoperiod(s);

-test design (e.g. chamber size, number of test chambers and replicates, number of embryos 

per replicates);

-method of preparation of stock solutions and frequency of renewal (the solubilising agent and 

its concentration must be given, when used);

-the nominal test concentrations, the means of the measured values and their standard 

deviations;

-dilution water characteristics: pH, hardness, temperature, dissolved oxygen concentration, 

residual chlorine levels (if measured), total organic carbon, suspended solids and other 

measurements made;

-water quality within test vessels, pH, temperature and dissolved oxygen concentration;

-detailed information on feeding (e.g. type of foods, source, amount given and frequency).

Results:

-Data for the control (plus solvent control when used) and the treatment groups as follows, 

embryological development, hatching (hatchability and time to hatch), posthatch survival, 

growth (total length and body weight), sexual differentiation (secondary sex characteristics 

and gonadal histology), reproduction (fecundity and fertility), and vitellogenin (VTG);

-Statistical analysis used and results of this.

LITERATURE

Kang IJ, Yokota H, Oshima Y, Tsuruda Y, Yamaguchi T, Maeda M, Imada N, Tadokoro H, 

Honjo T. 2002. Effects of 17β-estradiol on the reproduction of Japanese medaka (Oryzias 

latipes). Chemosphere 47:71–80.

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: (in press).

Seki M, Yokota H, Matsubara H, Maeda M, Tadokoro H, Kobayashi K. 2002. Fish full 

life-cycle testing of the weak estrogen 4-tert-pentylphenol in medaka (Oryzias latipes) 
16

Environ Toxicol Chem : (submitted).

U.S. Environmental Protection Agency. 1986. Fish life-cycle toxicity tests; Hazard Evaluation 

Division Standard Procedure EPA 540/9–86–137; Office of Pesticide Programs: Washington, 

USA.

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.

Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H, Honjo T, Kobayashi K. 2001a. 

Life-cycle toxicity of 4-nonylphenol to medaka (Oryzias latipes). Environ Toxicol Chem

20:2552–2560.
17

ANNEX 1

SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER

SUBSTANCE CONCENTRATION

Particular matter < 20 mg/L

Total organic carbon <2 mg/L

Unionized ammonia <1 μg/L

Residual chlorine <10 μg/L

Total organophosphorus pesticides <50 ng/L

Total organochlorine pesticides plus polychlorinated 

biphenyls

<50 ng/L

Total organic chlorine <25 ng/L
18

ANNEX 2

MMT Toxicant Sampling Guideline

The objective is to have an adequate number of samples to characterize the mean, standard 

deviation and range of exposure concentrations as well as intra-treatment variation. During the 

pretest period, sampling may have to be done quite frequently across and within treatments to 

establish diluter performance. Sampling and analysis of generated or mixed stock , mixing 

cells and delivery lines solutions, in addition to exposure tanks are usually necessary to 

calibrate a diluter system. During this pretest period, the concentration variability and stability 

of generated or mixed stock solutions and calibration standards can be observed.

To characterize the exposure treatments toxicant concentrations, weekly sampling is usually 

sufficient. This would include samples of two replicate tanks of each treatment of an assay 

that has up to six replicates per treatment. If the design is twelve replicates per treatment, 

sample three of the replicates each sampling period. Throughout the assay, sample different 

replicates of a treatment such that all replicates of a treatment have been measured at least 

once during a three to four week period. Every sample set includes one duplicate and 

fortified sample for every twelve exposure samples. External or internal calibration standards 

may be used as deemed appropriate. For a compound that does not behave in a reasonably 

predictable manner or is subject to biological degradation, it may be necessary to measure 

treatments more than once per week. Sampling more frequently is often necessary during 

the first few weeks of an assay until the toxicant behavior is understood or if frequent 

replacement of toxicant stock bottles is necessary.

The following is an example of a minimal sample set for a calibrated diluter with six 

treatments levels (five exposure and control) using six replicates / treatment. 

Samples: # of Samples

Treatment 1 Control A&D replicates 2

Treatment 2 Low Exposure A&D replicates 2

Treatment 3 Exposure A&D replicates 2

Treatment 4 Exposure A&D replicates 2

Treatment 5 Exposure A&D replicates 2

Treatment 6 High Exposure A&D replicates 2

Duplicate sample T3 A replicate 1

Fortified sample T3 + (spike) A replicate 1

____________

 Total # samples 14
19

Annex 3

Control Parameters

Growth- Weight and length measurements are taken for all fish sampled at 9 and 15 weeks 

post fertilization (wpf). Following this protocol will yield expected wet weights at 9 weeks 

wpf of 85 mg – 145 mg for males and 95 mg – 150 mg for females. The expected weights at 

15 wpf are 250 mg – 330 mg for males and 280 mg – 350 mg for females. While there may 

be substantial deviations from these ranges for individual fish, a replicate or treatment with 

mean weights substantially outside of these ranges would suggest problems with feeding, 

temperature control, water quality, disease or any combination of these factors.

Hatch- Hatching success in controls is typically around 90%, however, values as low as 80% 

are not uncommon. Hatch success less than 75% may indicate insufficient agitation of the 

developing eggs or inadequate care in handling the eggs such as lack of timely removal of 

dead eggs leading to fungal infestation of the eggs.

SurvivalThis endpoint is measured for both the F1 and F2 generations and is defined as the number of 

surviving fish from hatch until 3 wpf. This number is usually 90% or greater for controls but 

survival rates as low as 80% for controls are not alarming. Survival rates in controls of less 

than 80% would be cause for concern and may indicate insufficient cleaning of the aquaria 

leading to loss of larval fish through disease or from suffocation due to low dissolved oxygen 

levels. Mortality may also occur as a result of injury during tank cleaning and by the loss 

of larval fish to the drain system of the tank.

VitellogeninWhile absolute levels of vitellogenin, expressed as copies/ng of total RNA, may vary 

greatly between laboratories due to the procedures or instrumentation used, the ratio of 

vitellogenin should be around 200 times greater in control females versus control males. It is 

not uncommon for this ratio to be as high as from 1000 to 2000, however, ratios less than 200 

are suspect and may indicate problems with sample contamination or problems with the 

procedure and/or reagents used.

Secondary Sexual CharacteristicsThis endpoint is defined as the total number of segments in the fin-rays of the anal fin papillae. 

At 9 wpf, normal ranges for males will be 40-80 papillae and 0 for females. By 15 wpf, the 

range for control males should be about 80-120 and 0 for control females. For unexplained 

reasons, in rare instances some males will have no papillae present by 9 wpf but since all 

control males develop papillae by 15 wpf, this is most likely caused by delayed development. 

The presence of papillae in control females indicates the presence of XX males in the 

population.

XX-males –

The normal background incidence of XX males in culture appears to be about 4% at 250 C 
20

with the incidence increasing with increased temperature. Steps should be taken to minimize 

the proportion of XX males in the population. Since the incidence of XX males appears to 

have a genetic component and is therefore heritable, monitoring the culture stock and ensuring 

that XX males are not used to propagate the culture stock is an effective means to reduce the 

incidence of XX males in the population.

Spawning activitySpawning activity in the control replicates should be monitored daily prior to conducting the 

fecundity assessment . The control pairs can be qualitatively assessed visually for evidence of 

spawning activity. By12-14 wpf most control pairs should be spawning. Low numbers of 

spawning pairs by this time indicates potential problems with the health, maturity or 

well-being of the fish. 

FecundityHealthy, well fed 12-14 wpf medaka generally spawn daily, producing in the range of 15 to 50 

eggs per day. Mean daily egg production for all control spawning pairs should exceed 15 eggs 

per day and may reach as high as about 40 eggs per day. Less than this amount may indicate 

immature, malnourished or unhealthy spawning pairs.

FertilityThe percentage of fertile eggs for control spawning pairs is typically in the 90% range with 

values in the mid-to-upper 90s not uncommon. Fertility rates of less than 75% for control eggs 

are suspect and may indicate either unhealthy individuals or less than ideal culture conditions.