Overview of Aquatic Case Study

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Mysid Life Cycle Detailed Review
Paper NACEPT Endocrine Disruptor Methods
Validation Subcommittee July 2002 Leslie
Touart
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Detailed Review PaperMYSID LIFE CYCLE TOXICITY
TEST

• WORK PERFORMED BY
• On behalf of the United States Environmental
  Protection Agency
• EPA CONTRACT NUMBER 68-W-01-023

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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

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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

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METHODS USED IN THIS ANALYSIS

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

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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

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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.

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TEST SPECIES

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

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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

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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

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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

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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

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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

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Homesimysis costata

• 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

• Strengths
• Ecologically relevant to the Northeast Pacific
  region
• Large brood sizes
• Ease of handling and maintenance
• Used extensively
• Standardized protocol

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Mysidopsis intii

• Weaknesses
• Field-collected --identified prior to use
• Tests required to measure EDC-related endpoints
  must be developed
• Must be fed dietary supplement of copepods

• Strengths
• Ecologically important in northeast Pacific coast
• Shorter generation time than H. costata
• EPA sponsored 7-day toxicity testing protocol

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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

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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

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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

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CANDIDATE PROTOCOLS

• ASTM E1191 (ASTM 1997)
• OPPTS 850.1350 (EPA 1996)
• Chapman (1995)
• EPA-supported study (Langdon et al. 1996)

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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

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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

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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)
  lt40 Growth MSD lt50 ?g

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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 gt3
  average number of young/female

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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)

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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

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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

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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

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Questions

• Does the EDMVS agree that the two-generation
  method recommended with Americamysis bahia is
  appropriate?
• Does the EDMVS agree that prevalidation should
  evaluate the increased sensitivity of a
  two-generation design over the existing
  one-generation standard practice?
• Should EPA explore the feasibility and utility of
  biochemical endpoints, as described in the DRP,
  for possible addition to the recommended
  protocol?
• Does the EDMVS have suggestions to improve the
  DRP?