•J.-'lec Slates        Clfice of Water       EPA 505/8-90-OC6b
            Environmental Protection    (EN-336)         September 1990
x°/EPA      Culturing  Mysids
            (Mysidopsis bahia)

            Supplemental Report
            For Saltwater
            Video Training Tape

                                               EPA 505/8-90-006b

 U.S. Environmental Protection Agency
Office of Water Enforcement and Permits
           Permits Division
          401 M Street, SW
       Washington, DC  20460
    EPA Contract No. 68-C8-0015
                                                     Printed on Recycled Paper

      This report is a supplement to the video training tape "Culturing Mysidopsis bahitf (EPA, 1990a).  The
 techniques illustrated in the tape and described in this report are based on the methods used at EPA's
 Environmental Research Laboratory (ERL) in Narragansett, Rhode Island to culture Mysidopsis bahia
 (mysids) for use in marine toxicity tests. These test methods are published in the EPA manual  Short-Term
 Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine
 Organisms (EPA, 1988).

      This report and the video tape it accompanies are part of a series of training tapes and reports
 produced by EPA's Office of Water Enforcement and Permits. The tape entitled "Mysid (Mysidopsis bahia)
 Survival, Growth, and Fecundity Test" (EPA, 1990b) complements the material in this report by explaining
 the 7-day short-term chronic test method using mysids. • These tapes are available through The National
 Audiovisual Center, Capitol Heights, Maryland 20743. Other available tapes include the freshwater series:

      "Culturing Ceriodaphnia dubid'
      "Culturing Fathead Minnows (Pimephales promelas)
      " Ceriodaphnia Survival and Reproduction Toxicity Tests"
      "Fathead Minnow Larval Survival and Growth Toxicity  Test".

      This report has been funded wholly or in part by the Environmental Protection Agency under contract
68-C8-0015 to  Technical Resources, Inc.  It has been subject  to the Agency's review, and it has been
approved for distribution as an EPA document. Mention of trade names or commercial products does not
constitute  endorsement or recommendation for use.

     This report was produced for EPA's Office of Water Envorcement and Permits (Laura Phillips, Work
AssigomentManager) under contract to Technical Resources, Inc. (Lynn Bowler, Work Assignment
Manager).  Technical direction and expertise on the culture methods presented in this report were provided
by Suzanne Lussier, Randy Comeleo, and other staff of the EPA Environmental Research Laboratory in
Narragansett, Rhode Island. Several other individuals also have provided input to the techniques described
for culturing mysids, and while the list is  not inclusive, these are George Morrison (EPA, ERL-
Narragansett), Margarete Heber (EPA, Office of Water Enforcement and Permits), Bill Peltier (EPA,
Region 4, Athens, GA), and Richard Montgomery (TRJ).

      This report and the video "Culturing Mysidopsis bahia" (EPA, 1990a) were produced by EPA to clarify
 and expand on methods explained in the EPA manual Methods for Measuring the Acute Toxicity of Effluents
 to Freshwater and Marine Organisms (EPA, 1985). Healthy test animals and laboratory personnel who are
 familiar with the cuituring and handling procedures of the test animals are critical for accurate  and successful
 toxicity test results.

      Mysidopsis bahia, M. almyra, M, bigelowi, Metamysidopsis ilongata, and Neomysis americana, called
 mysids or opossum shrimp, have all been used in toxicity tests.  This report focuses on Mysidopsis bahia, the
 EPA-recommended species used in  the mysid survival, growth, and fecundity test (Method 1007; EPA, 1988).
      Mysidopsis bahia, are found in the coastal waters of the Gulf of Mexico (see Figure 1).  Mysids usually
 appear transparent with a yellow, brown, or black tint and range from 4.4 mm to 9.4 mm in length
 (Molenock, 1969).  Mysidopsis bahia differ from the other Mysidopsis species by the armature of the telson,
 the spines on the uropod, and an unsegmented antennal scale (Molenock, 1969).  These morphological
 characteristics used to identify Mysidopsis bahia are presented in Figure 2.

      The cuituring procedures presented in this report and illustrated in the video were  developed to meet
 the specific needs of the mysid in each of its life stages.  The first section of this report covers the selection
 and preparation of the water for cuituring and presents options for water delivery systems.  The  second
 section explains how to set up and maintain mysid cultures specifically for providing healthy test  animals.
 The third section provides details on the food preparation methods used at ERL-Narragansett. Instructions
 for collecting young of the same age for testing are given in the fourth section. A glossary is  provided
 describing some of the terms associated with cuituring mysids. Additional references providing more
 information on cuituring mysids are presented throughout the text and are summarized as Appendix A.
Appendix B provides a list of the apparatus and equipment needed to culture and test mysids.

                                                            dorsal process

 antennal scale
            8th thoracic limb    PleOP
                                           WATER AND LIGHT
 Culture Water

      A primary consideration when starting mysid cultures is the culture water. EPA recommends using
 natural seawater, although artificial sea salts also have been used.  If natural seawater is used, it must be
 contaminant-free and filtered through a 0.45 /im screen before use. The filter removes participates and
 possible predators.  The source of the culture water should be uncontaminated, consistent, reliable, and
 periodically checked to ensure these qualities.

      Although seawater is preferable, artificial seawater can be made by adding one of several
 recommended sea salts to distilled, deionized, or dechlorinated tap water.  Satisfactory brands used for mysid
 cultures include Forty Fathoms*, Hawaiin Marine Mix®, Environmental Systems Sea Salts*, Fritz Super
 Salts®,  and Steven Spotte's GP-2* formula (Lussier et al., 1988).  Reconstituted seawater must be aged
 approximately one month before it is used for cultures. Placing saltwater fish in the seawater helps hasten
 the aging process. The aged water should be tested with a small population of mysids before using it for
 cultures.  More specific instructions for the preparation of artificial seawater are listed in EPA's manual
 (EPA, 1985) or by commercial suppliers.

      The optimum water conditions for culturing mysids are presented in Table 1.


      For optimum growth and fecundity,  the photoperiod for mysid cultures should be 16  hours light and 8
hours dark.  ERL-Narragansett uses a system that turns the lights on and off gradually so as  not to startle
the mysids. The light intensity should be 50-100  foot-candles.

                 Table 1.  Recommended Culture Conditions for Mysidopsis bahitf



  Dissolved oxygen







  Tank Size


  Biological filter/algal mat



7.1 mg/1

0.1-03 mg/1

<0.05 mg/1

<20 mg/1

150 mg/1

12-hr light:12-hr dark to 16-hr light:8-hr dark

20 nm

10*55 gal

Dolomite, oyster shells, coral

Spirulina subsalsa
1  Source: Lussier et al., 1988-

 Culture Vessels

       Mysids can be cultured in tanks of various sizes.  The most commonly used are the 20 and 29 gallon
 aquaria.  Wider tanks are more suited for culturing than taller ones since a large surface area to volume
 ratio provides good oxygen exchange.  Tanks, as with all culturing equipment, should be cured in the culture
 water for approximately 3-5 days before being used for animals

 Water Delivery Systems

       Mysids can be cultured in flow-through, recirculating, or static systems. The preferred system is the
 flow-through arrangement where water is delivered to the tanks at a measured rate and the run off is
 discharged out of the system (see Figure 3). The flow rate  through the culture tanks should be no less than
 4-5 liters per hour or 2 turn overs per day.  Non-toxic materials such as glass, fiberglass, teflon, and
 polyvinylchloride (PVC) pipe  are recommended for the water delivery system.  Materials such as rubber,
 copper, brass, or plastic could become a source  of toxicity to the system and should be avoided.

      Recirculating systems can also be used to  culture mysids and should be designed to provide the same
 flow rate as the flow-through system.  However, recirculating systems must also provide a biofiltering system
 that can be constructed out of any non-toxic, high-surface-area material such as crushed coral, pea  gravel, or
 dolomite. A sand filter may also be added to the system.

      Static systems are made of a series of tanks that are independently filtered  and supplied with water.
The advantage of this type of system is that problems such  as disease are confined  to one tank and complete
culture crashes are less common.  Each tank in a static system should be supplied with an undergravel filter
and water changes should be made by replacing  one-half of the tank's volume of water with fresh culture
water every other day. Static systems are harder to maintain than flow-through or  recirculating systems due
to evaporation.  Tanks should be covered and care must be taken to avoid the concentration of salts as the
water evaporates.

               Figure 3.  Intermittent Flow^P^ugh Water Delivery System

                               CULTURE STAJRT UP AND MAINTENANCE
 Starting Cultures

      Once the  culture system and water source are designed, obtained, and seasoned, mysids can be
 purchased from a number of sources. A reliable supplier will certify that the correct species has been
 shipped.  EPA laboratories also will supply mysids depending on demand and availability. If test  animals are
 not needed immediately, cultures should be started with juveniles to allow laboratory personnel to become
 familiar with mysid handling and maintenance requirements before learning to collect the young.

      Mysids should be shipped  in Nalgene* containers packed inside coolers or polyfoam boxes within
 cardboard shipping cartons. The shipping density should be 100 mysids per liter and the container should
 have adequate airspace to ensure a supply of oxygen throughout the shipping period.  No food should be
 added to the containers.  A reliable overnight delivery service should be used for shipment.

      After the shipment is received the mysids must be acclimated to the receiving laboratory's culture
 water and conditions.  The temperature  and salinity of the water  used for shipping must be measured.  Slow
 adjustment of the water temperature can be accomplished by placing the container in  a water bath.  The
 salinity can be adjusted by adding new culture water to the water used for shipment. Increases or decreases
 in temperature  and salinity should not exceed 2-3° C or 2-3 %o, respectively, per day.

      For optimum growth and reproduction the stocking density for adult mysids should be between 200-300
per 20 gallon tank or approximately 20 mysids per liter.  A healthy, unstressed culture should have at least
70%  of the females carrying eggs in their brood pouch. Juveniles can be stocked at higher densities than


      Figure 4 shows the life cycle of a mysid.  Mysids produce live young called early juveniles. These
 juveniles are planktonic for the first 24 hours post release and then settle to the bottom where they orient to
 the current in the tank and begin to feed. Depending on water temperature and diet, females reach sexual
 maturity in 12-20 days.  Brood pouches appear at the age of 12-15 days and young are released at
 approximately 17-20 days. A gravid female is identified by an enlarged and darkened brood pouch containing
 the developing embryos.  The female  is ready to release the young when the eyespots can be identified in the
 brood pouch. Females average 5-7 young per brood, but can produce as many as 20 in one brood.  Multiple
 broods are produced for several months at a rate of one approximately every 4-6 days.
 Collecting  Test Animals

      To conduct toxicity tests using mysids, animals of the same age must be collected and pooled together.
 To accomplish this, gravid females are separated and their young are collected and held until the proper age
 for starting tests. For testing needs, assume a reproduction rate of two juveniles per female per day because
 not all females will release their young on the same day.

      At ERL-Narragansett brood chambers such as the one illustrated in Figure 5 are  used to collect test
 animals. Gravid females are collected from a minimum of three culture tanks and placed in a 4 liter
 Nalgene® beaker that is placed inside a separatory runnel containing culture water.  The solid plastic bottom
 of the Nalgene® beaker is replaced by 1 mm mesh screen. The screen allows the newly released young to
 pass through while preventing the adults from leaving the beaker.

      Once the females are placed in the brood chamber, provide food and gentle aeration or water  flow by
placing  an  airstone in the neck of the  separatory runnel or by providing water inflow and outflow to the
funnel.  The females should be left overnight and the young collected the next day.

      To harvest the young, remove the airstone and drain the separatory funnel into a 300 /jm mesh cup
placed in a culture dish. The mesh cup should be partially submerged in water to prevent injury to the
young.  While the water is draining, gently lift and dunk the beaker

              multiple broods
              for 2-3 months
 first brood release
    day 20
     DAY 16
2° sex characteristics
     day 12
                     DAY 10
  DAY 4
                           Figure 4. Life Cycle Of Mysidopsis bahia

                                               Separate ry
                                             Culture Dish
Figure 2.  Apparatus for collection of postlarval mysids from gravid females.
         From Lussier et al., 1988.

 containing the females to wash any remaining young out through the screen. As the water drains from the
 funnel, gently rinse the sides 2-3 times with clean seawater to wash out any mysids that may stick to the
 sides.  The females should be placed back into the culture tanks. The young can be used immediately for
 testing or grown out in a separate tank to the desired age for test start up.  The harvested young should be
 maintained at conditions similar to the regular cultures.

       An alternative system for collecting young is a siphon entrapment system, or a "mysid generator" (see
 Figure 6).  The  siphon inlet is covered by a 750 /im screen that excludes adults and allows juveniles to pass
 through to a collection vessel. In the collection vessel juveniles are deposited into a 350-370 /*m Nitex*
 screen cup. The juveniles in the screen cup are collected daily for test use. When using mysid generators,
 care must be taken to siphon all of the juveniles out of the tank each day.  Otherwise, the collected juveniles'
 ages may not be within 24 hours of each other as test methods require.

 Tank Cleaning

       Culture tanks should  be cleaned at least once each month. The sides of each tank should be scraped
 to remove any algal growth and the gravel should be stirred to dislodge the accumulated debris. This will
 clear the dolomite filter.

      Approximately twice each year, the tanks should be completely emptied and scrubbed. At this time the
 gravel should also be replaced.  It is important to  cure any new materials as described under Starting
 Cultures, above,  before using them in culture tanks.

 Record Keeping

      Culture tanks should be monitored and all conditions recorded on data sheets that are kept in a
 permanent file.  These record sheets can then be used to assess any problems that may occur with the
 cultures and assist in the elimination of possible causes.

      Figure 7 is  a data sheet adapted from the one used by ERL-Narragansett for mysid cultures. Each of
the conditions is  checked  daily and initialed by the technician taking the reading, checking the condition, or
performing the task.  Daily tasks performed are temperature, pH, salinity, and dissolved oxygen
measurements, seawater flow and air flow checks, and feeding (twice daily).

                        Pump Return
                                  — Culture Tank (75 L)
                       Screened Intake

           Post-larvae Trap

Collection Tank
                  Figure 6. A mysid generator. From Lussier et al., 1988.

                            MYSID CULTURE TANK CONDITIONS
                            Figure 7.  Data sheet for mysid cultures.

                            MYSID CULTURE TANK CONDITIONS
                            Figure 7. Data sheet for mysid cultures.

should be drained through a 150 pm screen and rinsed with clean seawater to remove any chemicals released
during hatching.

      To determine the correct amount'of Anemia for feeding, an aliquot of the hatched Anemia should be
counted under a microscope to determine the density of the culture.  This density will serve as a reference to
ensure that future cultures are hatching at the same rate.

      Once the Anemia are rinsed, the volume of clean seawater that is added determines the volume of
food provided to each tank. From the calculated and adjusted density of the diluted food supply, determine
the volume of food needed for each tank by estimating a feeding rate of 150 Anemia per mysid per day.
This should be supplied over two feedings and should be adjusted so that there are always live Anemia
available for the mysids to feed on.

Aitemia - Brine shrimp used as food for mysid cultures; Brazilian or Columbian strains are preferable.

Cyst - The life stage of unhatched Anemia.

Fecundity - Productivity or fertility.

Flow-through water delivery system - An open water flow system that delivers fresh water or seawater to
      culture tanks and is disposed of after it leaves those tanks.

Mysid (Mysidopsis bahia) - An estuarine crustacean ranging 4.4 mm to 9.4 mm in length found from the
      Gulf of Mexico and along the Atlantic coast used in test procedures as an indicator species for aquatic

Nauplii - The first stage of newly-hatched Aitemia.

Recirculating water delivery system - A water flow system that treats water after it passes through the
      culture tanks (usually with sand and biofilters) and delivers the same treated water back to the tanks.

Static water system - An enclosed system contained within one culture tank. The water is filtered through
      an underground or charcoal filter and is delivered back to the same tank.

                                              APPENDIX A

 American Society for Testing and Materials. 1987. Practice for using brine shrimp nauplii as food for
      testing animals in aquatic toxicology.  ASTM Designation E- 1203-87. Philadelphia, PA.

 Blatter, J.H.S., F.S. Russell, and M. Yonge, eds.  1980.  The biology of mysids and euphausiids. Part 1. The
      biology of the mysids. Adv. Mar. Biol. 18:1-319.

 Brattegard, T. 1969. Marine Biological Investigations in the Bahamas 10.  Mycidacea from shallow water in
      the Bahamas and southern Florida, Part 1.  Sarsia 39:17-106.

 Davey, E.W., J.H. Gentile, S J. Erickson, and P. Betzer.  1970. Removal of trace metals from marine culture
      media.  Limnol. Oceanogr. 15:486-488.

 Parrell, D.H.  1979. Guide to the Shallow-water mysids from Florida. Fla. Dept. Environ. Reg., Techn. Ser.
 Fbtheringham, N., and S.L. Brunenmeister.  1975.  Common marine invertebrates of the northwestern Gulf
      coast.  Gulf. Publ. Co., Houston, TX.

 Heard, R.W.  1982.  Guide to the common tidal marsh invertebrates of the northeastern Gulf of Mexico.
      Publ. No. MASGP-79-004, Mississippi- Alabama Sea Grant Consortium, Ocean Springs, MS.

 Johns, D.M., WJ. Berry, and W. Walton. 1981. International study on Artemia. XVI. Survival, growth, and
      reproductive potential of the mysid Mysidopsis bahia Molenock fed various geographical strains of the
      brine shrimp Artemia. J. Exp. Mar. Biol. Ecol. 53:209-219.

 Lawler, A.R. and S.L. Shepard.  1978.  Procedures for eradication of hydrozoan pests in closed-system mysid
      culture. Gulf Res. Rept.

 Lussier, S.M., A. Kuhn, MJ. Chammas, and J. Sewall.  1988. Techniques for the laboratory culture of
      Mysidopsis species (Crustacea: Mysidacea).  Environ. Tox. Chem.  7:969-977.

 Molenock, J. 1969.  Mysidopsis bahia, a new species of mysid (Crustacea: Mysidacea) from Galveston  Bay,
      Texas.  Tulane Stud. Tool. Bot. 15(3):113-116.

Nimmo, D.R. and T.L. Hamaker.  1982.  Mysids in toritity testing - a review. HydrobioL 93:171-178.

Nimmo, D.R., T.L. Hamaker, E. Matthews, and W.T. Young.  1982.  The long-term  effects of suspended
      participates on survival and reproduction of the mysid shrimp, Mysidopsis bahia, in the laboratory. In:
      G.F. Mayer, ed., Ecological Stress and the New York Bight:  Science and Management. Estuarine
      Res. Found., Columbia, S. Carolina, pp. 41-50.

 Nimmo, D.R., T.L. Hamaker, CA. Scanners. 1978. CuJturing the mysid (Mysidopsis bahid) in flowing sea
      water or a static system. In: Bioassay Procedures for the Ocean Disposal Permit Program, U.S.
      Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida.  EPA-
      600/9-78-010.  pp. 59-60.

 Price, W.W. 1982. Key to the shallow water Mysidacea of the Texas coast with notes on their ecology.
      Hydrobiol.  93(l/2):9-21.

 Stuck, K.C., H.M.  Perry, and R.W. Heard. 1979a. An annotated key to the Mysidacea of the North Central
      Gulf of Mexico.  Gulf Res. Rept. 6(3):225-238.

 Stuck, K.C., H.M.  Perry, and R.W. Heard. 1979b. Records and range extensions of Mysidacea from coastal
      and shelf water of the Eastern Gulf of Mexico.  Gulf Res. Rept. 6(3):239-248.

 U.S. EPA.  1985.  Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
      Organisms.  Third Edition.  Environ. Monit. and Supp. Lab., Cincinnati, OH. EPA/600/4-85/013.

 U.S. EPA.  1988.  Short-term Methods of Estimating the Chronic Toxicity of Effluents and Receiving Waters
      to Marine and Estuarine Organisms. Environ. Monit. and Supp. Lab., Cincinnati, OH. EPA/600/4-

 U.S. EPA.  1990a.  Culturing Mysidopsis bahia. Videotape. Produced by the Office of Water Enforcement
      and Permits. 19:13 min.

 U.S. EPA.  1990b.  Mysid  (Mysidopsis bahid) Survival, Growth, and Fecundity Test.  Videotape and
      Supplemental Report.  Office of Water Enforcement and Permits. 31:19 min.; 26 pp.
      EPA 505/8-90-006a.

 Venables, B. 1986. Mysidopsis sp.:  Life history and culture workshop report.  Gulf Breeze, FL. October
      15-16, 1986, Institute of Applied Sciences, North Texas State University, Denton, TX.

Ward, S.H.  1984.  A system for laboratory rearing of the mysid, Mysidopsis bahia Molenock.  Progr. Fish-
      Cult. 46(3):170-175.

Wigley, R.L. and B.R. Burns. 1971.  Distribution  and biology of mysids (Crustacea:  Mysidacea) from the
      Atlantic Coast of the United States in the NMFS Woods Hole Collection.  Fish Bull. 69:717-746.

                                               APPENDIX B

                                      APPARATUS AND EQUIPMENT
 Facilities for holding and acclimating test organisms.

 Brine shrimp culture unit — see Food Preparation above.

 Mysid culture unit - see Culture Start Up and Maintenance above.

 Samplers ~ automatic sampler, preferably with sample cooling capability, that can collect a 24-hour
       composite sample of 5 liters.

 Environmental chamber or equivalent facility with temperature control (26.+. TC).

 Water purification system -- Millipore Super-Q*, deionized water or equivalent.

 Balance -- capable of accurately weighing to 0.000001 g.

 Reference weights, Class S -- for checking performance of balance.

 Drying oven -- 105 °C, for drying organisms.

 Desiccator — for holding dried organisms.

 Air pump ~ for supplying air.

 Air line, and air stones -- for aerating cultures, brood chambers, and holding tanks, and supplying air to test
      solutions with low  DO.

 pH and DO meters -- for routine physical and chemical measurements.

 Tray ~ for test vessels; large enough to hold 8 vessels at one time.

 Standard or micro-Winkler apparatus --  for determining DO and checking DO meters.

 Dissecting microscope (240-400X magnification) — for examining organisms in the test vessels to determine
      their sex and to check for the presence of eggs in the oviducts of the females.

Light box — for  illuminating organisms during examination.

Refractometer or other method — for determining salinity

Thermometers, glass or electronic,  laboratory grade - for measuring water temperatures.

 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979) -- to
      calibrate laboratory thermometers.

 Test vessels - 200 ml borosilicate glass beakers or 8 oz disposable plastic cups (manufactured by Falcon
      Division of Becton, Dickinson Co., 1950 Williams Dr., Oxnard, CA 93030) or other similar containers.
      Cups must be rinsed thoroughly in distilled or deionized water and then pre-soaked (conditioned)
      overnight in dilution water before use. Forty-eight (48) test vessels are required for each test (eight
      replicates at each of five effluent concentrations and a control).

 Beakers or flasks - six, borosilicate glass or non-toxic plasticware, 2000 ml  for making test solutions.

 Wash bottles ~ for deionized water, for washing organisms from containers and for rinsing small glassware
      and instrument electrodes and probes.

 Volumetric flasks and graduate cylinders -  Class A, borosilicate glass or non-toxic plastic labware, 50-2000
      ml for making test solutions.

 Separatory funnels, 2-Iiters — 2 - 4 for culturing Anemia.

 Pipets, volumetric ~ Class A, 1-100 ml.

 Pipets, automatic -- adjustable, 1-100 ml.

 Pipets, serological ~ 1-10 ml, graduated.

 Pipet bulbs and filters - PROPIPET*, or equivalent.

 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for transferring organisms.

 Forceps (fine tips such as jewelers' forceps)  ~ for transferring organisms to weighing boats.

 NTTEX® mesh sieves (150 /im and 100 \aa) - for concentrating organisms.

Depression glass slides or depression spot plates - two, for observing organisms.