Mysid Life Cycle Detailed Review Paper

NACEPTEndocrine Disruptor Methods Validation

SubcommitteeJuly 2002

Leslie Touart

Detailed Review Paper:

MYSID LIFE CYCLE TOXICITY TEST

WORK PERFORMED BY

On behalf of the United States Environmental Protection Agency

EPA CONTRACT NUMBER 68-W-01-023

METHODS USED IN THIS ANALYSIS

• On-line Literature Search (August 15th 2001)• “Dialog” On-Line search with database Biosis

Previews Aquatic Science and Fisheries Abstracts • Endocrine disruptor screening methods for mysids

and sheepshead minnows• Key Words “sheepshead minnow” “mysid shrimp*

or reproduc* toxicity or devel*”• Approximately 526 records were refined down to

26 papers that were reviewed

METHODS USED IN THIS ANALYSIS

• Second search of Biosis and Aquatic Science and Fisheries abstracts was performed on August 22, 2001 which resulted in approximately 184 records

• Additional Search August 22 –24 on the ISI Web of Science database. Twenty references were found

METHODS USED IN THIS ANALYSIS

External/Internal Peer Review• Dr. Jerry Neff - Battelle• Jeff Ward -Battelle• EPA Technical Experts

OVERVIEW AND SCIENTIFIC BASIS OF MYSID LIFE CYCLE TOXICITY TEST

• Estuaries, which are important ecosystems, are among the earliest recipients of endocrine disrupting chemicals (EDCs)

• Crustaceans are often among the most abundant and most sensitive organisms (particularly mysids) in estuaries and they form vital links in food webs

• Many insecticides are considered putative EDCs. Certain insecticides formulated as IGRs adversely affect crustaceans by disrupting molting and metamorphosis

OVERVIEW AND SCIENTIFIC BASIS OF MYSID LIFE CYCLE TOXICITY TEST

• Endocrine system of an invertebrate differs from that of a vertebrate; therefore, the response of an invertebrate to an EDC could be expressed differently

• Ecdysteroids, which are the molting hormones in mysids, are also involved in the control of reproduction and embryogenesis. Mysids show promise as a potential indicator for evaluating ecdysteroid and EDC interaction.

TEST SPECIES

• Americamysis bahia• Holmesimysis costata• Mysidopsis intii• Neomysis integer

Americamysis bahia

• Reach 10 mm total length• Ecologically relevant to Gulf of Mexico

up through Narragansett, Rhode Island• Sexually mature at 12-20 days, brood

pouch fully formed at 15 days, developing young carried 2-5 days resulting in life cycle of between 17 to 20 days

• Females produce 11 juveniles/brood

Holmesimysis costata

• Reach 7 mm total length• Ecologically relevant to northeast Pacific

region• Sexually mature at 42 days, young are

released at about 65 to 73 days• Species are field-collected and available

year round

Mysidopsis intii

• Adults reach 7 mm total length• Ecologically relevant throughout South

America and Southern California• Sexually mature with young released at

about 20 days• Species are field-collected and then

easily cultured in the laboratory

Neomysis integer

• Females reach 18 mm total length, males are smaller

• Ecologically relevant throughout Northern Europe

• Sexually mature at 42 days, young are released at about 65 to 73 days

• Females can produce up to 80 juveniles/brood

• Species are field-collected and then easily cultured

Americamysis bahia

StrengthsWidely availableEasily culturedEasily identified from

others Short generation timeWidely usedStandardized protocol

WeaknessesMay not be ecologically relevant to colder-water materials-testing

Homesimysis costata

StrengthsEcologically relevant to

the Northeast Pacific region

Large brood sizesEase of handling and

maintenanceUsed extensivelyStandardized protocol

WeaknessesLonger generation

timeDifficulty in raising

multiple broodsField-collected --

identified prior to use

Tests required to measure EDC related endpoints must be developed

Mysidopsis intii

StrengthsEcologically important in

northeast Pacific coastShorter generation time than H.

costataEPA sponsored 7-day toxicity

testing protocol

WeaknessesField-collected --

identified prior to useTests required to

measure EDC-related endpoints must be developed

Must be fed dietary supplement of copepods

Neomysis integer

StrengthsEcologically important in

EuropeProtocols to measure

EDC- related endpoints are underway

WeaknessesField-collected --

identified prior to use

Tests required to measure EDC-related endpoints must be developed

ROUTES OF ADMINISTRATION OF

CHEMICAL EXPOSURE

• Aqueous Continuous flow-through systemConstant concentration of spiked water to the

test chamber

• SedimentMysids have been observed to collect

sediment, manipulate it at mouth region, and drop it. Therefore, clean spiked sediment could be used

• Dietary UptakeMix chemical with mysid food prior to feeding

POTENTIAL MEASUREMENT ENDPOINTS

• Survival• Molting frequency• Growth: ash-free dry weight and length • Measures of reproductive performance

– Sexual maturity– Time to first brood release– Total number of offspring

• Biochemical measures– Metabolic disruption– Vitellogenin induction– Cytochrome P450 enzyme Levels– Blood glucose levels

CANDIDATE PROTOCOLS

1) ASTM E1191 (ASTM 1997)2) OPPTS 850.1350 (EPA 1996)3) Chapman (1995)4) EPA-supported study (Langdon et al.

1996)

ASTM E 1191

Test Species: A. bahia, A. bigelowi, and A. almyra

Chemical Exposure:7 days after median brood release

Exposure Measurement Endpoints:Adult survival, body length, dry weight Number of young produced

Acceptance Criteria: 70% Survival of Adults75% of adult females produce young 3 average number of young/female

OPPTS 850.1350 Test Species: A. bahia

Chemical Exposure: 28 days

Exposure Measurement Endpoints:Adult survival, body length, dry weight and Number of young produced

Acceptance Criteria: 75% of parent females produce young 3 average number of young/female

CHAPMAN (1995)

Test Species:H. costata

Chemical Exposure:7 days

Exposure Measurement Endpoints:Adult survivalAdult growth

Acceptance Criteria: 75% of control survival 0.40 g dry weight in control Survival minimum significant difference (MSD) <40% Growth MSD <50 g

LANGDON (1996) Test Species: M. intii

Chemical Exposure: 28 days

Exposure Measurement Endpoints:Adult survival, body length, dry weightNumber of young produced

Acceptance Criteria: 75% of adult females produce young, and >3

average number of young/female

RECOMMENDED PROTOCOL-BLENDING OF EPA AND ASTM

Test Species: Americamysis bahia

Duration:Two-generation: 7 days after median first brood release in F1”

Reproductive Endpoints: Survival: (P, F1’, F1”, F2)Molt frequency: (P, F1’, F1”)Time to maturation: (P and F1”)Time to first brood release: (P and F1”)Time to second brood release: (P)Growth: length and dry weight: (P, F1’, F1”)Brood size/number of offspring: (P, and F1”)

RECOMMENDED PROTOCOL

Biochemical Endpoints:

Metabolic disruption: (P and F1”)

Steroid metabolism: (P and F1”)Vitellogenin induction: (P and F1”)Cytochrome P450 enzymes: (P and F1”)Blood glucose: (P and F1”)

Test Validity Criteria:

75% in parent controls

3 average number young/female/day Water quality requirements are met

SIGNIFICANT DATA GAPS

• Determine specific information on longer testing duration and on incorporating a second generation

• Biochemical Measurement Endpoints:– Refine steroid metabolism endpoints for mysids– Study cytochrome P540 enzyme level– Conduct vitellogenin mechanistic studies to confirm

endocrine disruption versus metabolic toxicity– Final goal: determine whether specific endpoint

responses can be linked to different classes of compounds affecting ecdysteroid, androgen, or other hormonal cycles

IMPLEMENTATION CONSIDERATIONS

• Prevalidation studies following the ICCVAM validation process Recommend studies to determine how

biochemical matrices may be related to hormonal disturbances

• Validation of the study design through interlaboratory comparisons

Questions1) Does the EDMVS agree that the two-generation

method recommended with Americamysis bahia is appropriate?

2) Does the EDMVS agree that prevalidation should evaluate the increased sensitivity of a two-generation design over the existing one-generation standard practice?

3) Should EPA explore the feasibility and utility of biochemical endpoints, as described in the DRP, for possible addition to the recommended protocol?

4) Does the EDMVS have suggestions to improve the DRP?