DRAFT: Medaka Reproduction Test

Guideline March 27, 2013

Medaka Reproduction

Test Guideline

1 April 20131 Medaka Reproduction Test Guideline

OBJECTIVE

This test g uideline 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 t he calculation of a No

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

mechanistic biomarker responses (namely, vitellogenin a nd gonad histology). The method is applicable to a variety of chemicals, i ncluding endocrine disrupters and general toxicants.

The medaka ( Oryzias latipes ) is th e 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 f i sh 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 p resent p rotocol 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 Inst itute 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 hav ing potential endocrine disrupto r activity ( e

. g

. androgenic or oestrogenic activity) then othe r 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 through out multiple generations of their life

- cycle. The data

ca

n be used to support T ier 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 F02 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 measure s the reproductive performance of medaka is started by exposing young reproductively active fish for 3 weeks, d uring 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 1 5 weeks post fertil ization (wpf). Fish from the F1 g eneration 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 pha se 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 t o 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. Fis h 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 l ess than 5% of population in seven days: accept the batch.3 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, SO 4 ), 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

a

nd mix the appropriate ratio of stock che mical solution with dilution water into a dilution cell ( aquarium ). These dilutions may be delivered to the various exposure aquarium s using multichannel peristaltic pumps. The flow rate of the stock solution should be calibrated in accordance with analyti cal 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 t he

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 t est s 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 concentr ation 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 provid es data for determining the lowest

- observed

- effect conce ntration and no

- observed

- effect concentration

, which are necessary for risk assessment . A minimum of six replicate t est 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 th e test. During4 the assessment of reproducti on, 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 17 β

- estradiol

.

Administration of t est 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 chos en 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

a

lso 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 solvent s 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 te st 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 carrie r 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 aquarium s 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 aquarium s. 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 t hese measurements are necessary to set up the exposure system, they will also

prov ide insight regarding t he aqueous s tability of the chemical

. After the exposure is initiated , it is important to monitor the c oncentration of the test

chemical in the exposure aquarium s frequently enough to allow accurate characterization of

the actual c oncentrations 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 treatme nt as

necessary. W eekly sampling of at least two replicate aquarium s from each treatment is usually5 sufficient to define the concentration of each treatment through time. O ne of the samples

should be taken from the same treatment re plicate at each sampling time and the other sample

should be systematically rotated f rom 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 b iological degradation in the dilution/exposure system, it may be necessary to measure treatments more than once per week. A suggested guideline for analytical sampling is presented in Annex 2.

PERFORMANCE OF THE TEST

Briefly, the test consists of con tinuously 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, t here are 6 treatments; five chemical concentration groups and a control group . The number of replicates w ithin each tre atment varies throughout the test. Initially, t here 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 . Th e measured endpoints i nclude 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 phenotyp e)

, histological evaluation of gonad al sex and inte rsex
, and finally, a histopathology evaluation of kidney, liver

, gon ad , 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 s uch 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 de pendable 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

tempe rature throughout the test is 25 .5 ° C, and th e photoperiod is 16h light and 8 h dark. The light intensity should be between 100

- 500 lux d uring the light period

, but may be increased up to 1,000 lux during test maintenance and sample collectio n. The control and exposure water flow to each 2 L aquarium is 20 ml/min. The general design employs 6 treatments including

controls h ere after 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

.6 Table 1. Test Conditions for the Proposed Medaka Reproduction Test (MRT)

Recommended species Medaka (Oryzias latipes)

Test type Continuous flow

- through

Water temperature 2 5 .5 ± 0.5 o C

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

Photoperiod 16 h light, 8 h dark

Loa ding rate F 0 - 2 fish/replicate; F 1 and F 2 - 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 F 0 - 11 weeks post

- fertilization, F 1 and F 2 continuously from fertilization

No. of organisms per replicate F 0 - 2 adults; F 1 and F 2 - 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 F 0

- 24 for each test concentration and 48 for controls; F 1 and F 2 - 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 O 2 /L.

Exposure period Trans

- generational test: F 0 from 11

- 15 wpf, F 1 and F 2 from 0

- 15 wpf

Primary endpoints Su rvival: F 1 from 1

- 3 wpf and from 3

- 15 wpf

Growth : length, weight, (F 1 a t 9 wpf and 15 wpf

Hatch: F 1 and F 2 only

Anal Fin Papillae: F 1 assessed a t 9 wpf and 15 wpf

Fecundity: F 1 assessed daily for 21 days, 12

- 14 wpf

Fertility: F 1 assessed daily for 21 days, 12

- 14 wpf

Vitellogenin: F 1 only at 9wpf7 Optional endpoints Time to first spawn for F 1 generation.

Test acceptability Dissolved oxygen > 60% of saturation; mean temperature of 2 5 .5 ± 0.5 o C; ≥ 70% survival in

control treatments of each generation; successful reproduc tion in at least 6 5% 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 te st chemical. The F0 fish are selected based on their age, from 11 to 15 wpf old, weight, at least 300 mg for female s and 250 mg for males, and having sexual genotype and phenotype s that match . While n o specific culturing proto col is re commended, appropriate husbandry practices should be

followed . A t the USEPA laboratory in Duluth, MN, medaka are reared in 2L aquarium s with

12 0 larval fish per aquarium until 4 wpf, and 10 fish per aquarium until 9 wpf . At this time breeding pairs are selected, base d on sexual phenotype, and placed in 2L aquarium s (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 pai rs 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 brief ly assess the breeding pair s for spawning

competency . Only competent f emale

- 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 aquarium s (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. T est f ish 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 colle cted during the first two days of s tud y w eek s 4 and 18 for initiat ing the F1 and F2 generations respectively. A fter 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 fro m 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 incubato rs 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 i ncubators are placed in the8 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 ha tch small numbers (maximum of 20 ) medaka eggs within the exposure system. The i ncubator 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

stainle ss 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 exchang e 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 8 th day post fertili zation, the aeration is stopped, causing the eggs to settle to the bottom of the incubator. This promotes hatching, typ ically over the next two days. For each treatment, hatchlings are counted, pool ed, and systematically distributed to each of the replicate aquaria

. Embryos that have not hatched by the 14 th day post fertilization

are considered non

- viable and discarded.

Fin Clipping and Determination of Genotypic Sex to Setup Breeding Pairs

Det ermination of genotypic sex via fin clips in the F1 generation is done in Study Week 12

.9 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, eac h 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 a n 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

presenc e 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 pair s . If there a re not enough XX

- XY pair s 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 pair s . If there are not enough XX and XY individuals

present within a n 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 i s determined and can be traced back to that fish

. All the data colle cted 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 histolog ically

- 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 recomme nded 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 co ncentrations 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 gen otype (XX or XY) and then10 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 pai r.

Sampling of Breeding Pairs: Adults

At Study Week 18 (15 wpf) the F1 adults are sacrifice d 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 end point 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

- h istory 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 Endocrine

mediated Non

- Endocrine

Mediated Direct 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) Y11 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 (F 0) 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 f emale

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

treatment levels includi ng 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 control s allowed to incubate. The eggs may be incubated in separate “incubator

aquar ia ” set up for each treatment or in the same replicate aquari a 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

. Stud y 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 replicat e aquarium within a specific treatment with no more than 12 hatchlings placed into12 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 surviv al of the larval fish to this point is recorded.

Th e 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 pair s per replicate are randomly established while the 8 (nominal) remaining fish per replicate a re sampled for the vario us 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 treat ment 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 analy zed for the endpoints in Table 2 , adults. Study Week 19: On Study Day 128, the aeration (agitation) of the incubating egg s is turned off to expedite hatching. The number of hatchlings are recorded to determine F2 hatch

success.13 Exposure and Endpoint TimelineGen 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 treatment s

2 blocks for control treatment s 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 statistica lly 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 F 1 generation.

As experience is gained with this test, additi onal biological performance criteria can be incorporated.

DATA REPORTING14 Statistical analysis

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

document Current Approaches in the Statistical Analysis of Ecotoxicit y 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 form al 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 signif icant 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 t oxicity 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, anore xia, 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 mus t 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

affe cted 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. An y 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, num ber 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, embryolog ical 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 17 β

- estradiol on the reproduction of Japanese medaka ( Oryzias latipes ). Chemosph ere 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 l ife

- 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 t he 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 Ch em 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 μ g/L

Residual chlorine <10 μ g/L Total organophosphorus pesticides <50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls <50 ng/L

Total organic chlorine <25 ng/L18 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 pe rformance. 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 stabi lity 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 replic ates 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 appro priate. 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 wee ks 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 Exposur e 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 1419 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 f eeding, 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 ag itation 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.

Survival

This endpoint is measured for both the F1 and F2 generations and is defined as the num ber 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 insuffi cient 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.

Vitellogenin

While 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 100 0 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 Characteristics

This 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 presenc e 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 25 0 C20 with the incidence increasing with increased temperature. Steps shou ld 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 cul ture stock is an effective means to reduce the incidence of XX males in the population.

Spawning activity

Spawning activity in the control replicates should be monitored daily prior to conducting the fecundity assessment . The control pairs can be qualitatively assessed vi sually for evidence of spawning activity. By 12

- 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.

Fecundity

Healthy, 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.

Fertility

The percentage of fertile eggs for control spaw n ing pairs is typically in the 90% range with

values in the mid

- to

- upper 90s not uncommon. Ferti lity rates of less than 75% for control eggs are suspect and may indicate either unhealthy individuals or less than ideal culture conditions.