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C:\Documents and Settings\Leel\Desktop\WA 2-15 DRP DRAFT DETAILED REVIEW PAPER ON MYSID LIFE CYCLE TOXICITY TEST EPA CONTRACT NUMBER 68-W-01-023 WORK ASSIGNMENT 2-15 July 2, 2002 Prepared for L. Greg Schweer WORK ASSIGNMENT MANAGER U.S. ENVIRONMENTAL PROTECTION AGENCY ENDOCRINE DISRUPTOR SCREENING PROGRAM WASHINGTON, D.C. BATTELLE 505 King Avenue Columbus, Ohio 43201 TABLE OF CONTENTS 1.0 EXECUTIVE SUMMARY ....................................................... 1 2.0 INTRODUCTION .............................................................. 2 2.1 DEVELOPING AND IMPLEMENTING THE ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP).......................................... 2 2.2 THE VALIDATION PROCESS............................................. 2 2.3 PURPOSE OF THE REVIEW ............................................. 3 2.4 METHODS USED IN THIS ANALYSIS...................................... 4 2.5 ACRONYMS AND ABBREVIATIONS ....................................... 5 3.0 OVERVIEW AND SCIENTIFIC BASIS OF MYSID LIFE CYCLE TOXICITY TEST ........... 6 3.1 ECDYSTEROID SENSITIVITY TO MEASURED ENDPOINTS ................... 9 4.0 CANDIDATE MYSID TEST SPECIES ............................................ 11 4.1 AMERICAMYSIS BAHIA ................................................ 12 4.1.1 Natural History ................................................... 12 4.1.2 Availability, Culture, and Handling .................................. 12 4.1.3 Strengths and Weaknesses ....................................... 13 4.2 HOLMESIMYSIS COSTATA ............................................. 13 4.2.1 Natural History ................................................. 13 4.2.2 Availability, Culture, and Handling .................................. 13 4.2.3 Strengths and Weaknesses ....................................... 14 4.3 MYSIDOPSIS INTII .................................................... 14 4.3.1 Natural History ................................................. 14 4.3.2 Availability, Culture, and Handling .................................. 14 4.3.3 Strengths and Weaknesses ....................................... 15 4.4 NEOMYSIS INTEGER .................................................. 15 4.4.1 Natural History ................................................. 15 4.4.2 Availability, Culture, and Handling .................................. 15 4.4.3 Strengths and Weaknesses ....................................... 16 4.5 OTHER MYSID SPECIES ............................................... 16 5.0 EXPOSURE PROTOCOLS FOR REPRODUCTIVE AND DEVELOPMENTAL TOXICITY TESTS............................................................ 16 5.1 ROUTE OF EXPOSURE ................................................ 17 5.1.1 Water......................................................... 17 5.1.2 Sediment...................................................... 19 5.2 CONCENTRATION SERIES............................................. 19 5.2.1 Statistical Considerations......................................... 20 6.0 DESCRIPTION OF ASSAY ENDPOINTS REFLECTIVE OF REPRODUCTIVE AND DEVELOPMENTAL IMPAIRMENT ............................................... 22 6.1 GROWTH, MORPHOLOGICAL, AND BEHAVIORAL ALTERATIONS............. 22 6.1.1 Growth........................................................ 23 6.1.2 Morphology .................................................... 24 6.1.3 Behavior ...................................................... 25 6.2 MEASURES OF REPRODUCTIVE PERFORMANCE ......................... 26 6.2.1 Sexual Maturity................................................. 26 6.2.2 Time to First Brood Release ....................................... 26 6.2.3 Egg Development Time .......................................... 27 6.2.4 Brood Size (Fecundity ............................................ 27 Battelle Draft Report i July 2, 2002 6.2.5 Intersexuality................................................... 27 6.3 BIOCHEMICAL MEASURES............................................. 28 6.3.1 Metabolic Disruption (O:N ratios)................................... 28 6.3.2 Steroid Metabolism.............................................. 28 6.3.3 Vitellogenin .................................................... 29 6.3.4 Cytochrome P450 Enzymes ....................................... 29 6.3.5 Blood Glucose Levels ............................................ 30 7.0 RESPONSE TO ECDYSTEROID AGONISTS AND ANTAGONISTS .................... 30 7.1 POSSIBLE GENDER DIFFERENCES IN RESPONSE TO ECDYSTEROIDS ....... 30 8.0 RESPONSE TO ANDROGENIC AGONISTS AND ANTAGONISTS ..................... 32 8.1 ENDPOINT SENSITIVITY............................................... 32 8.2 GENDER DIFFERENCES............................................... 33 9.0 RESPONSE TO OTHER HORMONAL DISTURBANCES ............................. 34 10.0 CANDIDATE PROTOCOLS.................................................... 36 10.1 ASTM E1191 STANDARD GUIDE FOR CONDUCTING LIFE CYCLE TOXICITY TESTS WITH SALTWATER MYSIDS (ASTM 1997) .................. 36 10.2 OPPTS TEST GUIDELINE 850.1350 MYSID CHRONIC TOXICITY TEST (EPA 1996) ..................................................... 37 10.3 OTHER PROTOCOLS.................................................. 37 11.0 RECOMMENDED PROTOCOL AND ADDITIONAL DATA NEEDS ..................... 43 11.1 PREFERRED TEST SPECIES ........................................... 43 11.2 DESCRIPTION OF THE METHOD........................................ 44 11.2.1 General Procedures and Equipment ................................ 44 11.2.2 Test Validity.................................................... 47 11.3 ENDPOINTS: APPROPRIATENESS AND PREFERRED METHODS FOR QUANTIFICATION..................................................... 48 11.3.1 Reproductive and Development Endpoints ............................ 48 11.3.2 Biochemical Endpoints ............................................ 49 11.4 EXPOSURE PROTOCOL ............................................... 50 11.5 RESULTS AND REPORTING............................................ 50 11.5.1 Interpretation of Results .......................................... 50 11.5.2 Reporting Requirements .......................................... 55 11.6 SIGNIFICANT DATA GAPS FOR PROTOCOL OPTIMIZATION ................. 56 11.7 RESEARCH NEEDS ................................................... 57 12.0 IMPLEMENTATION CONSIDERATIONS.......................................... 58 12.1 ANIMAL WELFARE.................................................... 59 12.2 RECOMMENDED EQUIPMENT AND CAPABILITY ........................... 59 12.3 TESTING WITH NON-NATIVE SPECIES ................................... 59 13.0 REFERENCES.............................................................. 60 APPENDIX A. LITERATURE SEARCH ................................................ A-1 APPENDIX B. EXPERT INTERVIEWS ................................................. B-1 Battelle Draft Report ii July 2, 2002 List of Tables Page Table 2-1 Acronyms and Abbreviations .............................................. 5 Table 5-1 Types of Water Delivery Systems ......................................... 19 Table 10-1 Recommended Mysid Life Cycle Toxicity Test Conditions ...................... 38 Table 10-2 Recommended Test and Holding Conditions for Holmesimysis costata and Mysidopsis intii ........................................................ 41 Table 11-1 Mysid Two Generation Toxicity Test Conditions Recommended for Conducting Tests of Potential Endocrine Disrupting Chemicals .................. 51 Table 11-2 Measurement of Effects of Three Classes of Hormones ........................ 57 List of Figures Figure 5-1 Diluter............................................................... 18 Figure 11-1. Conceptual Diagram of Measurements Endpoints and Estimated Corresponding Timeline for the Mysid Two Generation Test ........................................ 45 Battelle Draft Report iii July 2, 2002 DRAFT DETAILED REVIEW PAPER MYSID LIFE CYCLE TOXICITY TEST 1.0 EXECUTIVE SUMMARY Endocrine disruptors are any chemicals that are known or suspected to cause adverse endocrine effects in organisms or their progeny. Such chemicals have received increased attention over the past decade because of the potential harm they can do to wild and domestic animals and ultimately to humans. Therefore, Congress authorized the United States Environmental Protection Agency (EPA) to develop a program to screen a wide array of chemicals found in drinking-water sources and food to determine whether they possess estrogenic or other endocrine activity that could have disruptive endocrine effects in humans. The aim of this program is to develop a two-tiered approach: that is, a combination of in vitro and in vivo mammalian and ecotoxicological screens (Tier 1), and a set of in vivo tests (Tier 2) for identifying and characterizing endocrine effects of pesticides, industrial chemicals, and environmental contaminants. The organisms used in the screening and testing will represent a variety of taxonomic groups, such as marine and terrestrial invertebrates, fish, and mammals. The present detailed review paper fulfills one of the EPA’s objectives in its validation process, namely, to summarize, explain, and document the relevant principles, methods, and techniques for a two-generation reproductive/developmental toxicity test using an invertebrate species of mysid shrimp for evaluating effects of potential endocrine-disrupting chemicals. After reviewing the current literature, the report recommends an initial Tier 2 protocol and an organism that will best meet the needs for testing; and it identifies issues that could
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