# Presentation: 351039

## Mysid Life Cycle 

**Detailed Review Paper**
**NACEPT**
**Endocrine Disruptor Methods Validation Subcommittee**
**July 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

- _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

## 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

## 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

## 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

## 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

## 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

- ASTM E1191 (ASTM 1997)
- OPPTS 850.1350 (EPA 1996)
- Chapman (1995)
- 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 Adults
- 75% 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 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

## 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

## 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

## Questions

- <date/time>

- 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?