1. Mysid Life Cycle Detailed Review Paper NACEPT Endocrine Disruptor Methods Validation Subcommittee July 2002 Leslie Touart

2. 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

3. METHODS USED IN THIS ANALYSIS On-line Literature Search (August 15 th 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

4. 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

5. METHODS USED IN THIS ANALYSIS External/Internal Peer Review Dr. Jerry Neff - Battelle Jeff Ward -Battelle EPA Technical Experts

6. 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

7. 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.

8. TEST SPECIES Americamysis bahia Holmesimysis costata Mysidopsis intii Neomysis integer

9. 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

10. 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

11. 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

12. 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

13. Americamysis bahia Strengths Widely available Easily cultured Easily identified from others Short generation time Widely used Standardized protocol Weaknesses May not be ecologically relevant to colder- water materials- testing

14. Homesimysis costata Strengths Ecologically relevant to the Northeast Pacific region Large brood sizes Ease of handling and maintenance Used extensively Standardized protocol Weaknesses Longer generation time Difficulty in raising multiple broods Field-collected -- identified prior to use Tests required to measure EDC related endpoints must be developed

15. Mysidopsis intii Strengths Ecologically important in northeast Pacific coast Shorter generation time than H. costata EPA sponsored 7-day toxicity testing protocol Weaknesses Field-collected -- identified prior to use Tests required to measure EDC-related endpoints must be developed Must be fed dietary supplement of copepods

16. Neomysis integer Strengths Ecologically important in Europe Protocols to measure EDC- related endpoints are underway Weaknesses Field-collected -- identified prior to use Tests required to measure EDC- related endpoints must be developed

17. ROUTES OF ADMINISTRATION OF CHEMICAL EXPOSURE Aqueous Continuous flow-through system Constant concentration of spiked water to the test chamber Sediment Mysids have been observed to collect sediment, manipulate it at mouth region, and drop it. Therefore, clean spiked sediment could be used Dietary Uptake Mix chemical with mysid food prior to feeding

18. 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

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

20. 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 Adults  75% of adult females produce young  3 average number of young/female

21. 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

22. CHAPMAN (1995) Test Species: H. costata Chemical Exposure: 7 days Exposure Measurement Endpoints: Adult survival Adult growth Acceptance Criteria:  75% of control survival  0.40  g dry weight in control Survival minimum significant difference (MSD) <40% Growth MSD <50  g

23. LANGDON (1996) Test Species: M. intii Chemical Exposure: 28 days Exposure Measurement Endpoints: Adult survival, body length, dry weight Number of young produced Acceptance Criteria:  75% of adult females produce young, and >3 average number of young/female

24. 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”)

25. 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

26. 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

27. 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

28. Questions 1)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?