Whole Effluent Toxicity Training
          Video  Series
          Saltwater Series

U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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WHOLE EFFLUENT TOXICITY  TRAINING VIDEO SERIES  saltwater series
          Red Algal (Champia parvula)
          Sexual Reproduction
          Toxicity Tests
          Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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                            NOTICE

    The revision of this guide has been funded wholly or in part by the
Environmental Protection Agency under Contract EP-C-05-063. Mention of
trade names or commercial products does not constitute endorsement or
                     recommendation for use.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                  Supplement to Training Video
Foreword
       This guide serves as a supplement to the video "Red Algal (Champia parvula) Sexual Reproduction Toxicity
       Tests" (EPA, 2009). The methods illustrated in the video and described in this guide support the methods
       published in the U.S. Environmental Protection Agency's (EPA's) Short-term Methods for Estimating the
       Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA,
       2002a), referred to as the Saltwater Chronic Methods Manual. The video and this guide provide details on
       preparing for and conducting the test based on the expertise of personnel at the following EPA Office of
       Research and Development (ORD) laboratories:

           National Health and  Environmental Effects Research Laboratory (NHEERL) - Atlantic Ecology Division
           in Narragansett, Rhode Island

           NHEERL - Gulf Ecology Division in Gulf Breeze, Florida

           National Exposure Research Lab (NERL) - Ecological Exposure  Research Division (EERD) in
           Cincinnati, Ohio

       This guide and its accompanying video are part of a series of training videos produced by EPA's Office of
       Wastewater Management. This Saltwater Series includes the following videos and guides:

           "Mysid (/Americamys/s bahia) Survival, Growth, and Fecundity Toxicity Tests"

           "Culturing Amer/camys/s bahia"

           "Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia punctulata"

           "Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests"

           "Sheepshead Minnow (Cyprinodon  variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
               and Growth Toxicity Tests"

       The Freshwater Series, released in 2006, includes the following videos and guides:

           "Ceriodaphnia Survival and Reproduction Toxicity Tests"

           "Culturing of Fathead Minnows (Pimephales promelas)"

           "Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests"

       All of these videos are available through the National Service Center for Environmental Publications
       (NSCEP) at 800 490-9198 or nscep@bps-lmit.com.

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U.S. ENVIRONMENTAL PROTECTION AGENCY                   Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                            Supplement to Training Video
                                             Intentionally Left Blank

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U.S. ENVIRONMENTAL PROTECTION AGENCY              Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                          Supplement to Training Video
   CONTENTS
   Foreword	i
   Introduction	1
   Background	1
   Culturing Champia parvula	1
   Culture Water	1
   Photoperiod	2
   Culture Vessels	2
   Preparing Algae for Testing	2
   Conducting the Test	3
   Collecting the Algae	3
   Effluent Preparation	3
   The Exposure Period	5
   The Recovery Period	6
   Terminating the Test	6
   Test Acceptability and Data Review	9
   Citations and Recommended References 	9
   Glossary	Glossary-1
   Appendix A: Nutrients and Media	A-l
   Appendix B: Apparatus and Equipment	B-l
   Appendix C: Reagents and Consumable Materials	C-l
   Appendix D: Summary of Test Conditions and Test Acceptability Criteria for the Red Macroalga,
   Champ/a parvula, Sexual Reproduction Test With Effluents and Receiving Waters	D-l

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U.S. ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
 ,                                                                        Supplement to Training Video
  FIGURES
  Figure 1. Life History of the Red Macroalga, Champia parvula. Left: Size and Degree of Branching
  in Female Branch Tips Used For Toxicity Tests [[[ ................... 2
  Figure 2. Apex of Branch of Female Plant, Showing Sterile Hairs and Reproductive Hairs
  (Trichogynes)  [[[ . ........... 3
  Figure 3. A Portion of the Male Thallus Showing Spermatial Sori. The Sorus Areas Are Generally
  Slightly Thicker and Somewhat Lighter in Color [[[ 3
  Figure 4. A Magnified Portion of a Spermatial Sorus. Note the Rows of Cells that Protrude from the
  Thallus Surface [[[ . [[[ 4
  Figure 5. Apex of a Branch on a Mature  Female Plant That Was Exposed To Spermatia from a Male
  Plant [[[ 4
  Figure 6. Receiving Water Data Form for the Red Macroalga, Champia parvula, Sexual
  Reproduction Test [[[ 5
  Figure 7. A Mature Cystocarp  [[[ 6
  Figure 8. Comparison of a Very Young Branch and an Immature Cystocarp ..................................... 7

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    U.S. ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                  Supplement to Training Video
 ^jfcsH

Introduction

       This guide accompanies the Environmental Protection Agency's (EPA's) video training for conducting red
       algal (Champia parvula) sexual reproduction toxicity tests (EPA, 2009). The test method is found in Section
       16 of EPA's Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
       Marine and Estuarine Organisms, Third Edition (EPA, 2002a). The test was developed by EPA's Office of
       Research and Development's (ORD's) National Health and Environmental Effects Research Laboratory-
       Atlantic Ecology Division (NHEERL-AED) in Narragansett, Rhode Island. The material presented in both the
       video and this guide summarizes the methods but does not replace a thorough review and understanding
       of the methods by laboratory personnel before conducting the test.
Background
       Under the National Pollutant Discharge Elimination System (NPDES) program (Section 402 of the Clean
       Water Act), EPA uses toxicity tests to monitor and evaluate effluents for their toxicity to biota and their
       impact on receiving waters. By determining acceptable or safe concentrations for toxicants discharged
       into receiving waters, EPA can establish NPDES permit limitations for toxicity. These Whole effluent toxic-
       ity (WET) permit limitations regulate pollutant discharges on a whole effluent effect basis rather than by a
       chemical-specific approach only.

       Whole effluent toxicity methods measure the synergistic, antagonistic, and additive effects of all the chemi-
       cal, physical, and additive components of an effluent that adversely affect the physiological and biochemi-
       cal functions of the test organisms. Therefore, healthy organisms and correct laboratory procedures are
       essential for valid test results. Laboratory personnel should be very familiar with the test methods and with
       red algae handling techniques before conducting a test.

       This supplemental guide covers the procedures for conducting the test according to EPA's promulgated
       methods (40 CFR Part 136; EPA, 2002c) and also provides some helpful information that is not presented
       in the Saltwater Chronic Methods Manual (EPA, 2002a).

       This guide summarizes methods developed at NHEERL-AED for estimating the chronic toxicity of marine
       or estuarine effluents and receiving waters on the sexual reproduction of the marine macroalga, Champia
       parvula. Males and females are exposed to effluents or receiving waters for 2 days, followed by a 5- to
       7-day recovery period for the female plants in a control medium. Cystocarp production by the female, which
       indicates sexual reproduction, is used as the endpoint. The test results determine the effluent concentra-
       tion causing a statistically significant reduction in the  number of cystocarps formed.

       This guide and accompanying video describe how the test is set up, initiated, terminated, and reviewed,
       including suggestions on maintaining healthy cultures of test organisms.
Culturing Champia parvula
       There are three macroscopic stages in the life history of Champia. The adult plant body (thallus) is hollow,
       septate, and highly branched. Only the mature male and female plants are used in toxicity testing. Mature
       plants are illustrated in Figure 1.

       To keep a constant supply of plant material available, maintain several unialgal stock cultures of males
       and females simultaneously. Also, new cultures should be started weekly from excised branches so that
       cultures are available in different stages of development.

       CULTURE WATER
       Natural seawater, or a 50-50 mixture of natural and artificial seawater, makes optimal culturing media.
       Seawater for cultures is filtered at least to 0.45 urn to remove most particulates and autoclaved for 30
       minutes at 15 psi (120C). Carbon stripping the seawater may be necessary before autoclavingto enhance
                                                                                                     I

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                  Red Algal (Chomp/a parvula) Sexual Reproduction Toxicity Tests
                                                                                Supplement to Training Video
                               Figure 1. Life History of the Red Macroalga, C/iamp/a parvula. Left: Size
                               and Degree of Branching in Female Branch Tips Used For Toxicity Tests
                                                                                        spermatia
                                                                                         fertilization
                               tetrasporangia
                                    TETRASP'
                                   Source: EPA 1987.
                                                                                        cystocarp
                                                                    5 mm
its water quality (EPA, 1990).
Instructions for carbon
stripping are provided in the
Saltwater Chronic Methods
Manual (EPA, 2002a). Nutrients
should be added to the water
to ensure healthy cultures.
Recipes for the culturing
medium and nutrient solutions
are provided in Appendix A. The
water temperature should be
maintained at 23C + 1C and
the salinity at 30%0  2%0

Gently aerate the cultures.
Change alternate cultures'
medium every week so that if a
stock solution should become
contaminated, the entire batch
will not be lost. While replen-
ishing the medium, divide the growing algae in half with sharp forceps or discard half of the biomass to prevent
overcrowding. New cultures also can be started at this time using 1 cm branch tips. Add nutrients using a pipet;
NHEERL-AED has found a squeeze bottle is quick and easy to use. At the end of approximately three weeks, there
should be enough plant material to conduct the test.

PHOTOPERIOD
The culture conditions should include a photoperiod 16 hours of light and 8 hours of darkness. The light
level should not exceed 500 ft-candles (75 uE/m2/s) and may have to be adjusted to that level, depending
on the reflecting characteristics of the incubators.

CULTURE VESSELS
Maintain stock cultures of males and females in separate, aerated, 1 L Erlenmeyer flasks containing 800
mL of the culture medium. All glass must be acid-stripped in 15 percent HCI and rinsed in deionized water
before use because some detergent residues  can be toxic to the Champia. At least every 6 months, the
glass should be cleaned to remove organic materials that can build up on the surface. Always use sterile
techniques when culturing the algae (i.e., autoclave all stock solutions and flame all tools before cutting or
transferring plants) to guard against microalgal contamination.

PREPARING ALGAE FOR TESTING
Examine the stock cultures to determine their readiness for testing. Place a few female branch tips  in
seawater in a petri dish, and examine them under a compound microscope to determine if trichogynes are
present. An inverted scope works best with the petri dishes, although standard slides and microscopes also
can be used.  Trichogynes are the short, fine reproductive hairs to which the spermatia attach (see Figure
2). They should be seen easily near the apex of the branch tip. Although both sterile hairs and trichogynes
occur on the apex, sterile hairs occur over the entire plant thallus. Sterile hairs are wider and generally much
longer than trichogynes, and appear hollow, except at their tip, where they seem to be plugged.

Males should be visibly producing spermatia.  Sometimes, the presence of spermatia sori can be deter-
mined by placing some male tissue in a petri dish and  holding it against a dark background. Mature sori
can be easily identified under a microscope along the edge of the thallus. The sorus areas are generally
thicker and lighter in color than the rest of the plant body. At higher magnification, the spermatia them-
selves can be seen (see Figures 3 and 4).

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                      Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                   Supplement to Training Video
       The readiness of the male stock
       culture can also be assessed
       by placing a portion of a female
       plant into a portion of the solu-
       tion from the male culture for
       a few seconds. Under a micro-
       scope, numerous spermatia
       should be seen attached to the
       sterile hairs and trichogynes of
       the female plant (see Figure 5).

       Once readiness is established
       for both males and females, the
       test can begin.

Conducting  the Test

       COLLECTING THE ALGAE
       Prepare cuttings from the most
       healthy-looking plants. Prepare
       the female cuttings first to mini-
       mize the chances of contaminat-
       ing them with water containing
       spermatia from the male stock
       cultures. Place each plant in
Figure 2. Apex of Branch of Female Plant, Showing Sterile Hairs and
Reproductive Hairs (Trichogynes)
                                     sterile hairs
                                                 ichogynes
Sterile hairs are wider and generally much longer than trichogynes, and appear hollow
except at the tip. Roth types of hairs occur on the entire circumference of the thallus, but
are seen easiest at the "edges." Receptive trichogynes occur only near the branch tips.

    Source: EPA 1987.
       a petri dish containing a small amount of seawater. Using a fine-point forceps or scalpel, prepare five cut-
       tings from the female plants for each treatment replicate, severing the plant 7-10 mm from the ends of the
       branch. Try to be consistent in the degree of branching in the cuttings,  since cystocarps form at the branch
       tips.

       For male plants, use one cutting for each treatment replicate, severing the plant about 2 - 3 cm from the
       end of the branch. If there are few branches, or the spermatial sori appear sparse, larger male cuttings may
       be needed. The cuttings can be kept at room temperature for up to an hour.
       EFFLUENT PREPARATION
       Effluent sampling should be
       conducted according to Section
       8 of the Saltwater Chronic
       Methods Manual (EPA, 2002a)
       and any specific requirements of
       a NPDES permit. The effluent or
       receiving waters should be held
       at 0C - 6C until used for test-
       ing. Under the NPDES program,
       lapsed time from sample collec-
       tion to first use in the test must
       not exceed 36 hours. Under
       special conditions or variances,
       samples may be held longer but
       should never be used for testing
       if held for more than 72 hours.
Figure 3. A Portion of the Male Thallus Showing Spermatial Sori. The
Sorus Areas Are Generally Slightly Thicker and Somewhat Lighter in
Color
             1 cm
                                            spermatial sorus
                                     Source: EPA 1987.

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                       Red Algal (Champla parvula) Sexual Reproduction Toxlcity Tests
                                                                                      Supplement to Training Video
Dilution Water
The type of dilution water used
to make the test concentrations
is dependent on the objectives
of the test Any specific require-
ments included in NPDES permits
should be followed.  The Saltwater
Chronic Methods Manual (Section
7) provides the following guidelines:
*  If the test is conducted to esti-
mate the absolute chronic tox-
ic/ty of the effluent, synthetic
dilution water should be used. If
the cultures were maintained in
different water than used for dilu-
tion water, a second set of control
replicates should be conducted
using the culture water.
  If the test is conducted to
estimate the chronic toxicity
of the effluent in uncon-
taminated receiving waters,
the test can be conducted using
a grab sample of the receiving
waters collected outside the influ-
ence of the outfall, other uncon-
taminated waters,  or standard
dilution water with the same
salinity as  the receiving waters. If
the cultures were maintained in
different water than used for dilu-
tion water,  a second set of control
replicates should be conducted
using the culture water,
  If the test  is conducted to
estimate the additive or miti-
gating effects of the effluent
on already contaminated
receiving waters, the test
must be conducted using receiv-
ing waters collected outside the
influence of the outfall.  Controls
should be conducted using both
receiving water and culture water.
Figure 4. A Magnified Portion of a Spermatial Sorus. Note the Rows
of Cells that Protrude from the Thallus Surface
                                  cuticle
                                               thallus surface
Source: EPA 1987.
Figure 5.      of a         on a                Plant That
         To            from a
                                              spermatia
The sterile hairs and trichogynes are covered with spermatia. Note that
few or no spermatia are attached to the older hairs  (those more than
I  mm from the apex).
Source: EPA 1987.

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 U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                  Red Algal (Champ/a parvula) Sexual Reproduction Toxicity Tests
                                                                               Supplement to Training Video
   Maintain the salinity of the test samples to 30%o  2%o. To do this, effluent samples may need to be
   adjusted using hypersaline brine (HSB). A recipe for MSB is provided in Appendix A of this manual.

   Approximately 1 hour before the test is to begin, adjust approximately 1 L of effluent to the test tempera-
   ture of 23C  1C and maintain that temperature while preparing the test concentrations. To test a series
   of decreasing concentrations of effluent, use a dilution factor of > 0.5. When starting with effluent that has
   0%0 salinity, the maximum effluent concentration that can be prepared at 30%o is 70 percent effluent. A
   table for preparing the samples is provided in Appendix A.

   THE EXPOSURE PERIOD
   A 125 ml Erlenmeyer flask is used for each test chamber, but any clean container can be used. The test
   chambers should be labeled using colored tape and marking pens to identify each treatment and replicate.
   These should be placed in randomized positions for the duration of the test.

   Under a hood, prepare five dilutions  using a > 0.5 dilution factor in 300 or 400 ml replicates.
   Approximately 1800 ml of effluent is required for a test conducted using a 0.5 dilution factor. This allows
   for enough of each prepared effluent concentration to provide four replicates at 100 ml and 400 ml for
   chemical analyses and water quality data. Record the water quality data on a form such  as the one pro-
   vided in Figure 6.
Figure 6. Receiving Water Data Form for the Red Macroalga, Champ/a parvula, Sexual Reproduction Test.
Site:
Collection Date:
Test Date:

Locations











Initial
Salinity











Final
Salinity












Source of Salts for Salinity Adjustment1











1Natural seawater, GP2 brine, GP2 salts, etc. (include some indication of amount.)
Source: EPA, 2002a.

   The 2-day exposure period starts when the algae are added to the test chambers. Add five female branch-
   es and one male branch to each prepared chamber. Pick up the branch at the base or cut end to avoid
   injuring the tips. The effluent must be in the test chamber before the algae are added.

   Cover the chambers with aluminum foil or a foam stopper, exposing the cultures to 16 hours of cool white
   light and 8 hours of darkness each day for the 2-day exposure, as well as the 5- to 7-day recovery peri-
   ods. Maintain the temperature at 23C + 1C, and the salinity between 28%o and 32%o with the variance
   between chambers on any day maintained at < 2%o.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                 Red Algal (Champia porvulo) Sexual Reproduction Toxicity Tests
                                                                             Supplement to Training Video
   Check on the chambers twice a day, and gently hand-swirl the chambers, or shake continuously at 100
   rpm on a rotary shaker. Spermatia are not motile, so some motion is critical during the exposure period for
   reproduction to occur. If desired, the media can be changed after 24 hours. Record the temperature daily
   from a thermometer placed in a flask of water among the chambers.
                                Routine chemical and physical observations should be made during the
                                test. Dissolved oxygen (DO) is measured at the beginning and end of
                                each 24-hour exposure period in one test chamber at each concentra-
                                tion and in the control. Temperature, pH, and salinity are measured at the
                                end of each 24-hour exposure period, also in one test chamber at each
                                concentration and in the control. Temperature also should be monitored
                                continuously, observed and recorded daily for at least two locations in the
                                environmental control system or the samples. The locations for determin-
   ing temperature should be sufficient to indicate any temperature variations in the environmental chamber.
pH should be measured in
the effluent sample before
any new test solutions are
made to determine changes
in the effluent sample.
   THE  RECOVERY PERIOD
   Prepare recovery bottles by labeling clean 100 - 200 ml vessels with the effluent concentrations tested,
   and fill them with 150 ml of natural seawater and nutrients. Smaller volumes can be used but may require
   changes of the medium to allow for adequate growth.

   After the 48-hour exposure period, use forceps to gently remove all of the females from each test chamber,
   and place them into recovery bottles. When all the replicates have been transferred, place the vessels
   under cool white light and aerate or shake for the 5- to 7-day recovery period. Aeration will enhance the
   growth rate of plants in the recovery bottles, although adequate growth will occur using a shaker. Aerate
   using plastic tubes held in place by foam stoppers.

   TERMINATING THE TEST
   At the end of the recovery period, drain the chambers and remove the females with forceps, starting with
   the control plants and ending with those in the highest concentration. Place the female plants between the
   inverted  halves of a petri dish      Figure 7. A Mature Cystocarp
   containing a small amount
   of seawater, and count the
   cystocarps under a stereo-
   microscope. Cystocarps are                      /. '  . '.*&.                 ^  . ^ostiole
   distinguished from young
   branches by the darkly                             ^i^^Uf fK.    ^!:*J^I J **	sPres
   pigmented spores enclosed
   in the nodule, and the apical
   opening for spore release
   (ostiole). Figures 7 through 9
   provide illustrations to help
   identify cystocarps.
                                  In the controls and lower effluent concentrations, cystocarps often occur in clusters of
                                  10 or 12.
                                  Source: EPA 1987.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                        Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                          Supplement to Training Video
                                        Figure 8. Comparison of a Very Young Branch and an Immature
                                        Cystocarp
                                                                            young branch
                                                                                     cells
                                                                                            immature
                                                                                            cystocarp
                                       Both the young branch and immature cystocarp can have sterile hairs. Trichogynes
                                       might or might not be present on a young branch, but are never present on an imma-
                                       ture cystocarp. Young branches are more pointed at the apex and are made up of
                                       larger cells than immature cystocarps, and never have ostioles.
                                       Source: EPA 1987.
                                       Figure 9. An Aborted Cystocarp.
                                       A new branch will eventually develop at the apex.
                                       Source: EPA 1987.

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 U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                   Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                 Supplement to Training Video
   If there is doubt about the identification of an immature cystocarp, aerate the plants a little longer in the
   recovery bottles. Within 24 to 48 hours, the suspected cystocarp will look more like a mature cystocarp
   or a young branch, or will have changed very little, if at all, indicating an aborted cystocarp. Occasionally
   cystocarps will abort, and these should not be included  in the counts. Aborted cystocarps are easily identi-
   fied by their dark pigmentation and/or by the formation of a new branch at the apex. Dead plants lose their
   pigmentation and appear white.

   Record all counts for the  test on a form such as the one provided in Figure 10.

Figure 10. Cystocarp Data Sheet for the Red Macroalga,  Champia parvula, Sexual Reproduction Test
   Collection Date:
   Exposure Began (date):
   Effluent or Toxicant:
Recovery Began (date):
Counted (date): 	
                            Treatment (% Effluent, mg/L, or receiving water sites)
Replicates

A 1
2
3
4
Mean

B 1
2
3
4
Mean

C 1
2
3
4
Mean

D 1
2
3
4
 Mean

Overall
Mean
Control




























































































































































































   Temperature:.
   Salinity:	
   Light:	:
   Source of Dilution Water:
Source: EPA, 1987.

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    U'S' ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                 Supplement to Training Video
Test Acceptability and  Data  Review
       Test data are reviewed to verify that EPA's WET test methods' test acceptability criteria (TAG) requirements
       for a valid test have been met. The algal sexual reproduction test requires that several criteria be met
       before the test results are considered acceptable.

              Control plants should average 10 or more cystocarps per plant and survival in the control must be
               80 percent or greater.

              Control and lowest-concentration exposed algae should be in good physical conditionfor exam-
               ple, the branches should not be fragmented. Broken or fragmented branches could indicate that
               the plants were unhealthy or stressed from the beginning of the test.

              The results from the replicate control chambers should be similar.

              All replicates from the affected concentration chambers should show effect.

       The concentration-response relationship generated for each multi-concentration test must be reviewed to
       ensure that calculated test results are  interpreted appropriately. In conjunction with this requirement, EPA
       has provided recommended guidance for concentration-response relationship review (EPA, 2000b).

       EPA's promulgated toxicity testing method manuals (EPA, 2002a, b) recommend the use of point estima-
       tion technique approaches for calculating endpoints for effluent toxicity tests under the NPDES program.
       The promulgated methods also require a data review of toxicity data and concentration-response data, and
       require calculating the percent minimum significant difference (PMSD) when point estimation (e.g., LC50,
       IC25) analyses are not used. EPA specifies the PMSD must be calculated when NPDES permits require sub-
       lethal hypothesis testing. EPA also requires that variability criteria be applied as a test review step when
       NPDES permits require sub-lethal hypothesis testing endpoints (i.e., no observed  effect concentration
       [NOEC] or lowest observed effect concentration [LOEC]) and the effluent has been determined to have no
       toxicity at the permitted receiving water concentration (EPA, 2002b). This reduces the within-test variabil-
       ity and increases statistical sensitivity when test endpoints  are expressed using hypothesis testing rather
       than the preferred  point estimation techniques.


Citations and Recommended References

       EPA. 1979. Methods for chemical analysis of water and wastes.  Environmental Monitoring and Support
               Laboratory, U.S. EPA, Cincinnati, OH 45268. EPA-600/4- 79/020,  revised March 1983.

       EPA. 1985. Aquatic Toxicity Testing Seminar Manual. 1985. National  Health and  Environmental Effects
               Research Laboratory-Aquatic Ecology Division, Narragansett, Rl. NHEERL-AED Contribution No.
               796.

       EPA. 1987. Guidance manual for conducting sexual reproduction test with the marine macroalga Champia
               parvula for use in testing complex effluents. Contribution No. X103. Thursby, G.B. and R.L. Steele.
               In: Schimmel S.C., ed. Users guide to the conduct and interpretation of complex effluent toxicity
               tests at estuarine/marine sites. Environmental Research Laboratory, U.S. EPA, Narragansett, Rl
               02882. Contribution No. 796. 265 pp.

       EPA. 1989. Biomonitoring for Control of Toxicity in Effluent  Discharges to the Marine Environment.  1989.
               U.S. EPA Center for Environmental Research Information, Cincinnati, OH; U.S. EPA Environmental
               Research Laboratory, Narragansett, Rl.  EPA/625/8-89/015.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY                Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                 Supplement to Training Video
                                                                                    ',$ , ,-, .ft .-
       EPA. 1990. Supplemental methods and status reports for short-term saltwater toxicity tests.
              G. Morrison and G. Chapman. ERL Contrib. No. 1199. Environmental Research Laboratory,
              U.S. EPA, Narragansett, Rl 02882. 127 pp.

       EPA. 1991. Technical Support Document for Water Quality-based Toxics Control. U.S. EPA Office of Water
              Enforcement and Permits, Washington, D.C. EPA-505-2-90-001.

       EPA. 2000a. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR
              Part 136). Office of Water, Washington, D.C. EPA 821-B-00-004.

       EPA. 2000b. Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications
              Under the National Pollutant Discharge Elimination System Program. Office of Wastewater
              Management, Washington, D.C. EPA 833-R-00-003.

       EPA. 2002a. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
              Marine and Estuarine Organisms, Third Edition. (Saltwater Chronic Methods Manual). Office of
              Water, Cincinnati, OH. EPA-821-R-02-014.

       EPA. 2002b. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
              and Marine Organisms, Fifth Edition. (Acute Methods Manual). Office of Water, Cincinnati, OH.
              EPA-821-R-02-012.

       EPA. 2002c. Final Rule. 40  CFR Part 136. Guidelines Establishing Test Procedures for the Analysis of
              Pollutants; Whole Effluent Toxicity Test Methods. 67 FR 69952-69972, November 19, 2002.

       EPA, 2009. Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests. Supplement to Training Video.
              Whole Effluent Toxicity Training Video Series, Saltwater Series. March 2009.
              EPA 833-C-09-001.

       Spotte, S., G. Adams, and P.M. Bubucis. 1984. GP2 as an artificial seawater for culture or maintenance of
              marine organisms. Zool. Biol. 3:229-240.

       EPA references are available online at www.epa.gov/npdes.

       If you need additional copies of this document, you can download  it at:
              www.epa.gov/npdes/wqbasedpermitting.
10

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yjg| U.S. ENVIRONMENTAL PROTECTION AGENCY                Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
Va'                                                                                Supplement to Training Video
Glossary
        Acute toxicity. An adverse effect measured on a group of test organisms during a short-term exposure in
               a short period of time (96 hours or less in toxicity tests). The effect can be measured in lethality
               or any variety of effects.

        Champia parvula. The scientific name for red algae. Champia parvula have soft, gelatinous, pinkish red,
               much-branched fronds that are densely matted, with blunt apices, to 100 mm high. Their axes are
               segmented, with nodal diaphragms. The segments are about as broad as long, filled with a watery
               mucilage. Red algae are found epiphytic on smaller algae in lower intertidal pools. They are found
               widely distributed in the Atlantic and Pacific marine environments.

        Chronic toxicity. An adverse effect that occurs over a long exposure period. The effect can be lethality,
               impaired growth, reduced reproduction, etc.

        Diluent water. Dilution water used to prepare the effluent concentrations.

        Effluent concentrations.  Concentrations or dilutions of an effluent sample to which test organisms are
               exposed to determine the biological effects of the sample on the test organism.

        Effluent sample. A representative collection of the discharge that is to be tested.

        Hypothesis testing. Technique (e.g., Dunnett's test) that determines what concentration is statistically
               different from the control. Endpoints determined from hypothesis testing are NOEC and LOEC.

        IC25 (Inhibition Concentration, 25%).  The point estimate of the toxicant concentration that would cause a
               25% reduction in a non-quantal biological measurement (e.g., reproduction or growth) calculated
               from a continuous model.

        LC50 (Lethal Concentration, 50%). The concentration of toxicant or effluent that would cause death to
               50% of the test organisms at a specific time of observations (e.g., 96-hour LC50).

        Lowest Observed Effect Concentration (LOEC). The LOEC is the lowest concentration of toxicant to
               which organisms are exposed  in a test, which causes statistically significant adverse effects on
               the test organisms (i.e., where the values for the observed endpoints are statistically significantly
               different from'the control). The definitions of NOEC and LOEC assume a strict dose-response
               relationship between toxicant  concentration and organism response.

        Minimum Significant Difference (MSD).  The MSD is the magnitude of difference from the control where
               the null hypothesis is rejected in a statistical test comparing a treatment with a control. MSD
               is based on the number of replicates, control performance and power of the test. MSD is often
               measured as a percent and referred to as PMSD.

        No Observed Effect Concentration (NOEC).  The NOEC is the highest tested concentration of toxicant to
               which organisms are exposed  in a full life-cycle or  partial life-cycle (short-term) test, that causes
               no observable adverse effect on the test organism (i.e., the highest concentration of toxicant
               at which the values for the observed responses are not statistically significantly different from
               the controls). NOECs calculated by hypothesis testing are dependent upon the concentrations
               selected.

        NPDES (National Pollutant Discharge Elimination System) Program. The  national program for issuing,
               modifying, revoking and reissuing, terminating, monitoring and enforcing permits, and imposing
               and enforcing pretreatment requirements, under Sections 307, 318, 402, and 405 of the Clean
               Water Act.

                                                                                              Glossary-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY                Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                     Supplement to Training Video
        Point Estimation Techniques. This technique is used to determine the effluent concentration at which
               adverse effects (e.g., fertilization, growth or survival) occurred, such as Probit, Interpolation
               Method, Spearman-Karber. For example, a concentration at which a 25% reduction in
               reproduction and survival occurred.

        Receiving Water Concentration (RWC). The RWC is the concentration of a toxicant or the parameter
               toxicity in the receiving water (i.e., riverine, lake, reservoir, estuary.or ocean) after mixing.

        Toxicity test. A test to  measure the toxicity of a chemical or effluent using living organisms. The test
               measures the degree of response of an exposed organism to a specific chemical or effluent.

        WET (Whole effluent toxicity). The total toxic effect of an effluent measured directly with a toxicity test.
Glossary-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                     Red Algal (Chompia parvula) Sexual Reproduction Toxicity Tests
                                                                                  Supplement to Training Video
Appendix A:
Nutrients and  Media
       The following instructions for nutrients are provided in the Saltwater Chronic Methods Manual
       (EPA, 2002a). Table A-l lists the additional nutrients to be added to natural or artificial seawater for stock
       cultures and test media. The concentrated stock solution is autoclaved at standard temperature and pres-
       sure for 15 minutes before the vitamins are added. Adjust the solution to about pH 2 before autoclavingto
       minimize the possibility of precipitation.

    Table A-l. Nutrient Stock Solution
Nutrient Stock
Solution"
NaNO3
NaH2PO4  H2O
Na2EDTA  2 H2O
Na3C6H507  2 H2O
lronb
Vitamins0
Amount IL Concentrated Nutrient Stock Solution
Stock Solution for
Culture Medium
6.35 g
0.64 g
133 mg
51 mg
9.75 ml
10 mL
Stock Solution for
Test Medium
l.58g
0.16 g

12.8 mg
2.4 mL
2.5mL
    3 Add 10 mL of appropriate nutrient stock solution per liter of culture or test medium.
    b A stock solution of iron is made that contains 1 mg iron/mL. Ferrous or ferric chloride can be used.
    c A vitamin stock solution is made by dissolving 4.88 g thiamine HCI, 2.5 mg biotin, and 2.5 mg Bi2 in 500 mL deionized
    water. Adjust vitamin stock to approximately pH 4, divide into 10 mL subsamples, and autoclave for 2 minutes before it is
    added to the nutrient stock solution.

Preparing Hypersaline Brine  (HSB)

       BACKGROUND
       Champia parvula cannot be cultured in 100% artificial seawater. However, 100% artificial seawater can be
       used during the 2-day exposure period. This allows 100% effluent to be tested.

       Salinity adjustments are a vital part of using marine and estuarine species for toxicity testing. The major-
       ity of industrial and sewage treatment effluents entering marine and estuarine waters contain little or no
       measurable salts. Therefore, the salinity of these effluents must be adjusted before exposing estuarine or
       marine plants and  animals to the solutions. The salinity of the effluent can be adjusted by adding HSB pre-
       pared from natural seawater (100%o), concentrated (triple strength) salt solution (GP2 described in table
       below), or dry GP2  salts (also below). Adjust the salinity of the effluent to 30%o. Control solutions should be
       prepared with the same percentage of natural seawater and at the same salinity as the effluent solutions.

       Constant salinity should be maintained across all treatments throughout the test for quality control.
       Matching the test solutions' salinity to the expected receiving water's salinity may require salinity adjust-
       ments. EPA NHEERL-AED uses HSB, prepared from filtered natural seawater, to adjust exposure solution
       salinities.

       HSB has several advantages over artificial sea salts that make it more suitable for use in toxicity testing.
       Concentrated brine derived from natural seawater contains the necessary trace metals, biogenic colloids,
       and some of the'microbial components necessary for adequate growth, survival, and/or reproduction of
       test organisms. It may be held for prolonged periods without any apparent degradation. Brine may be
       added directly to the effluent to increase the salinity, or may be used as control water by diluting to the
                                                                                                  A-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
Red Algal (Champ/a parvula) Sexual Reproduction Toxicity Tests
                              Supplement to Training Video
                                       ,'.- A. TvJVSa
       desired salinity with deionized water. The brine can be made from any high quality, filtered seawater supply
       through simple heating and aerating.

    Table A-2. GP2 Artificial Seawater for Use in Conjunction with Natural Seawater for the Red Macroalga,
    Champ/a parvula, Sexual Reproduction Toxicity Test
Compound
NaCI
Na2SO4
KCI
KBr
Na2B4O7- IOH20
MgCI2  6 H2O
CaCI2  2 H2O
SrCI2  6 H2O
NaHCO3a
Concentration (gIL)
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
Amount (g) Required for 20-L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
    The original formulation calls for autoclaving anhydrous and hydrated salts separately to avoid precipitation. However, if
    the sodium bicarbonate is autoclaved separately (dry), all of the other salts can be autoclaved together. Since no nutrients
    are added until needed, autoclaving is not critical for effluent testing. To minimize microalgal contamination, the artificial
    seawater should be autoclaved when used for stock cultures. Autoclaving (120C) should be for at least 10 minutes for
    1-L volumes, and 20 minutes for 10- to 20-L volumes.
    Artificial seawater should be prepared in 10- to 20-L batches. Effluent salinity adjustment to 30%o can be made by adding
    the appropriate amount of dry salts from this formulation, by using a triple-strength brine prepared from this formulation,
    or by using a 100%o salinity brine prepared from natural seawater.
    Nutrients listed in Table A-l should be added to the artificial seawater in the same concentration described for natural
    seawater.
    3 A stock solution of 68 mg/mL sodium bicarbonate is prepared by autoclaving it as a dry powder, and then dissolving it in
    sterile deionized water. For each liter- of GP2, use 2.5 mL of this stock solution.
    Source: EPA, 2002a. Modified from Spotte et a/., 1984. Constituents salts and concentrations were taken from EPA
    1990.

       GENERATING THE BRINE
       The ideal container for making brine from natural seawater has a high surface-to-volume ratio, is made of a
       non-corrosive  material, and is easily cleaned. Shallow fiberglass tanks are ideal.

       Collect high-quality (and  preferably high-salinity) seawater on an incoming tide to minimize the possibility
       of contamination. Special care should be used to prevent any toxic materials from coming in contact with
       the seawater. The water should be filtered to at least 10 |jm before placing into the brine tank. Thoroughly
       clean the tank, aeration supply tube, heater, and any other materials  that will be in direct contact with the
       brine before adding seawater to the tank. Use a good-quality biodegradable detergent, followed by several
       thorough deionized-water rinses. Fill the tank with seawater, and slowly increase the temperature to 40C.
       If a heater is immersed directly into the seawater, make sure that the heater components will not corrode
       or leach  any substances that would contaminate the brine. A thermostatically controlled heat exchanger
       made from fiberglass works well.

       Aeration prevents temperature  stratification and increases the rate of evaporation. Use an oil-free air
       compressor to prevent contamination. Evaporate the water for several  days, checking daily (or more or less
       often, depending on the  volume being generated) to ensure that the salinity does not exceed 100%o and   '
       the temperature does not exceed 40C. If these changes are exceeded, irreversible changes in the brine's
       properties may occur. One such change noted in original studies at ERL-N was a reduction in the alkalinity
A-2

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 U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                  Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                               Supplement to Training Video
   of seawater made from brine with salinity greater than 100%o, and a resulting reduction in the animals'
   general health. Additional seawater may be added to the brine to produce the volume of brine desired.

   When the desired volume and salinity of brine is prepared, filter the brine through a 10-um filter and pump
   or pour it directly into portable containers (5-gallon cubitainers or polycarbonate water cooler jugs are most
   suitable). Cap the containers.'and record the measured salinity and the date the brine was generated.
   Store the brine in the dark at room temperature until used.

   SALINITY ADJUSTMENTS USING MSB
   To calculate the volume of brine (Vb) to add to 0%o sample to produce a solution at certain salinity (Sf), use
   this equation:
    Where  Vb =
                                        vb  * sb = sf * vf
                volume of brine, ml
        Sb =    salinity of brine, %o
        Sf =    final salinity, %o
        Vf =    final volume, mL (brine brought to this volume with 0 %o sample).

Table A-3 gives volumes needed to make 30%o test solutions from effluent (0%o), deionized water, and
100%o MSB. At 30%o salinity, the highest achievable concentration is 70% effluent.
Table A-3. Preparation of Test Solutions at a Salinity of 30%o Using HSB for a Final Test Concentration
Volume of 1000 ml.
Exposure
Concentration (%)
70
25
7
2.5
0.7
Control
Effluent
(0%o)
(mL)
700
250
70
25
7

Deionized Water
(mL)

450
630
675
693
1,000
Hypersaline Brine
(100 %o)
(mL)
300
300
300
300
300

                                                                                                A-3

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A-4

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    U.S. ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Chomp/a parvula) Sexual Reproduction Toxicity Tests
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Appendix  B:
Apparatus and  Equipment
       Air lines, and air stones. For aerating cultures, brood chambers, and holding tanks, and supplying air to
               test solutions with low DO.

       Air pump. For oil-free air supply.

       Balance. Analytical, capable of accurately weighing to 0.00001 g.

       Beakers, Class A. Borosilicate glass or non-toxic plasticware, 1000 ml for making test solutions.

       Bottles. Borosilicate glass or disposable polystyrene cups (200 - 400 ml) for use as recovery vessels.

       Compound microscope. For examining the condition of plants.

       Count register. 2-place for recording cystocarp counts.

       Dissecting (stereomicroscope) microscope. For counting cystocarps.

       Drying oven. To dry glassware.

       Erlenmeyer flasks, 250 mL, or 200 ml disposable polystyrene cups, with covers. For use as exposure
               chambers.

       Environmental chamber or equivalent facility with temperature control (23  1C).

       Facilities for holding and acclimating test organisms.

       Filtering apparatus. For use with membrane filters (47  mm).

       Forceps, fine-point, stainless steel. For cutting and handling branch tips.

       Laboratory red macroalga, Champ/a parvula, culture unit. To test effluent or receiving water toxicity,
               sufficient number of sexually mature male and female plants must be available.

       Meters: pH and DO, and specific conductivity. For routine physical and chemical measurements.

       Micropipettors, digital, 200 and 1000 uL. To make dilutions.

       Pipet bulbs and filters. Propipet, or equivalent.

       Pipets, automatic. Adjustable 1 - 100 ml.

       Pipets, serological. 1-10 ml, graduated.

       Pipets, volumetric. Class A, 1 -  100 mL.

       Reference weights, Class S.  For checking performance of balance.

       Refractometer or other method. For determining salinity.

       Rotary shaker. For incubating exposure chambers (hand-swirling twice a day can be substituted).
                                                                                                B-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY                Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                   Supplement to Training Video
       Samplers. Automatic samplers, preferably with sample cooling capability, that can collect a 24-hour
               composite sample of 1 L.

       Thermometers. National Bureau of Standards Certified (see EPA 2002a). Used to calibrate laboratory
               thermometers.

       Thermometers. Bulb-thermograph or electronic-chart type for continuously recording temperature.

       Thermometers, glass or electronic, laboratory grade. For measuring water temperatures.

       Water purification system. Millipore Milli-Q deionized water or equivalent.

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

       Volumetric flasks and graduated cylinders. Class A, borosilicate glass or non-toxic plastic labware,
               10 - 1000 ml for making test solutions.
B-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY               Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                                                  Supplement to Training Video
     *>


Appendix C:

Reagents and  Consumable  Materials

       Aluminum foil, foam stoppers, or other closures.  To cover cultures and test flasks.

       Artificial seawater. A slightly modified version of the GP2 medium (Spotte, et al., 1984) has been used
               successfully to perform the red macroalga sexual reproduction test. The preparation of artificial
               seawater (GP2) is described in Table A-2.

       Buffers pH 4, pH 7, and pH 10. (Or as per instructions of instrument manufacturer) for standards and
               calibration check.

       Data sheets (one set per test). For data recording (see Figures 6 and 10).

       Disposable tips for micropipettors.

       Effluent, receiving water, and dilution water. Test waters, including effluent, receiving, and dilution
               water should be analyzed to ensure its quality prior to using in tests. Dilution water containing
               organisms that might prey upon or otherwise interfere with the test organisms should be filtered
               through a fine mesh (with 150 urn or smaller openings).

       Laboratory quality assurance samples and standards. For the above methods.

       Markers, waterproof.  For marking containers, etc.

       Mature red macroalga, Champia parvula, plants.

       Petri dishes, polystyrene. To hold plants for cystocarp counts and to cut branch tips. Other suitable
               containers may be used.

       pH buffers pH 4, pH 7, and pH 10. (Or as per instructions of instrument manufacturer) for standards and
               calibration check.

       Reagent water. Distilled or deionized water that does not contain substances which are toxic to the test
               organisms.

       Reference toxicant solutions. Reference toxicants such as sodium chloride (NaCI), potassium chloride
               (KCI), cadmium chloride (CdCI2), copper sulfate (CuS04), sodium dodecyl sulfate (SDS), and
               potassium dichromate (K2Cr207), are suitable for use in the NPDES Program and other Agency
               programs requiring aquatic toxicity tests.

       Saline test and dilution factor. The use of natural  seawater is recommended for this test.  A recipe for the
               nutrients that must be added to the natural seawater is given in Table A-l. The salinity of the test
               water must be 30%o and vary no more than  2%o among the replicates. If effluent and receiving
               water tests are conducted concurrently, the salinity of these tests should be similar.

               The overwhelming majority of industrial and sewage treatment effluents entering marine and
               estuarine systems contain little or no measurable salts. Therefore, exposure of the red macroalga,
               Champia parvula, to effluents will usually require adjustments in the salinity of the test solutions.
               Although the red macroalga, Champia parvula, cannot be cultured  in 100% artificial seawater,
               100% artificial seawater  can be used during the 2-day exposure period. This allows 100%
               effluent to be tested. It is important to maintain a constant salinity across all treatments. The
               salinity of the effluent can be adjusted by adding MSB prepared from natural seawater (100%o),
               concentrated (triple strength) salt solution  (GP2 described in Table A-2), or dry GP2 salts (Table

                                                                                                  C-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY                Red Algal (Champ/a parvula) Sexual Reproduction Toxicity Tests
                                                                                       Supplement to Training Video
                                                                                                 -; *  ,v -   rt <,* tSsSS-

                A-2), to the effluent to provide a salinity of 30%o. Control solutions should be prepared with the
                same percentage of natural seawater and at the same salinity (using deionized water adjusted
                with dry salts, or brine) as used for the effluent dilutions.

        Sample containers. For sample shipment and storage.

        Tape, colored.  For labeling test chambers.
C-2

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                   Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
                                                      Supplement to Training Video
Appendix D:
Summary of Test Conditions and Test Acceptability
Criteria for the Red Macroalga, Champia parvula, Sexual
Reproduction Test With Effluents and Receiving Waters
     (Note: this test is not listed at 40 CFR Part 136 for nationwide use)
Test type
Salinity
Temperature (C)
Light source
Light intensity
Photoperiod
Test chamber size
Test solution volume
Number of organisms per test chamber
Number of replicates per concentration
Number of organisms per concentration
Aeration
Dilution water
Test concentrations
Receiving waters
Dilution factor
Test duration
Endpoints
Test acceptability criteria
Sampling requirements
Sample volume required
Static, non-renewal (required)
30%o  2 %o of the selected test salinity (recommended)
23C  IC (recommended)
Cool-white fluorescent lights (recommended)
About 75 |jE/m2/s (500 ft-c) (recommended)
16 hr light, 8 hr dark (recommended)
200 mL polystyrene cups (with covers) or 250 mL Erlenmeyer flasks
(recommended)
100 ml (minimum required)
5 female branch tips and one male plant (recommended)
4 (3 required minimum)
24 (18 required minimum)
None; chambers are either shaken at 100 rpm on a rotary shaker or .
hand-swirled twice a day
30%o salinity natural seawater, or a combination of 50% of 30%o salinity
natural seawater and 50% of 30%o salinity GP2 artificial seawater
Effluents: 5 and a control (required minimum)
100% receiving water (or minimum of 5) and a control (recommended)
Effluents: ^ 0.5 (recommended)
Receiving Waters: None or S 0.5 (recommended)
2-day exposure to effluent followed by 7-day recovery period in control
medium for cystocarp development (required)
Reduction in cystocarp production compared to controls (required)
80% or greater survival, and an average of 10 cystocarps per plant in
controls (required)
For on-site tests, one sample collected at test initiation, and used within
24 hr of the time it is removed from the sampling device.
For off-site test, holding time must not exceed 36 hr before test use.
(required)
2 L per test (recommended)
  Source: EPA, 2002a. Saltwater Chronic Methods Manual,
                                                                 D-l

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D-2

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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WHOLE EFFLUENT TOXICITY  TRAINING VIDEO SERIES  saltwater series
           Culturing Americamysis  bahia
           Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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                      NOTICE

 The revision of this guide has been funded wholly or in part by the
  Environmental Protection Agency under Contract EP-C-05-063.
Mention of trade names or commercial products does not constitute
           endorsement or recommendation for use.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                Culturing Americomys/s bahia
                                                                                  Supplement to Training Video
Foreword
       This guide serves as a supplement to the video "Culturing Americamys/s bahia" (EPA, 2009a). The meth-
       ods illustrated in the video and described in this supplemental guide support the methods published in
       the U.S. Environmental Protection Agency's (EPA's) Methods for Measuring the Acute Toxicity of Effluents
       and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition (2002a), referred to as the Acute
       Methods Manual. The video and this guide provide details on culturing of mysids for the use in conducting
       tests based on the expertise of personnel at the following EPA Office of Research and  Development (ORD)
       laboratories:

           National Health and Environmental Effects Research Laboratory (NHEERL) - Atlantic Ecology Division
           in Narragansett, Rhode Island

           NHEERL - Gulf Ecology Division in Gulf Breeze, Florida

           National Exposure Research Lab (NERL) - Ecological Exposure Research Division (EERD) in
           Cincinnati, Ohio

       This guide and its accompanying video are part of a series of training videos produced by EPA's Office of
       Wastewater Management.  The video entitled "Mysid (Americamys/s bahia) Survival, Growth, and Fecundity
       Toxicity Tests" (EPA 2009b) complements the material in this video by explaining the 7-day short-term
       chronic toxicity test method using mysids. This Saltwater Series includes the following videos and guides:

           "Mysid (Americamys/s bahia) Survival, Growth, and Fecundity Toxicity Tests"

           "Culturing Americamys/s bahia"

           "Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia punctulata"

           "Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests"

           "Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
           and Growth Toxicity Tests"

       The Freshwater Series, released in 2006, includes the following videos and supplemental guides:

           "Ceriodaphnia Survival and Reproduction Toxicity Tests"

           "Culturing of Fathead  Minnows (Pimephales promelas)"

           "Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests"

       All of these videos are available through the National Service Center for Environmental Publications
       (NSCEP) at 800 490-9198 or nscep@bps-lmit.com.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                          Culturing Americamysis bahia
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    U'S" ENVIRONMENTAL PROTECTION AGENCY
                                                                          Culturing Americomysis bahia
                                                                             Supplement to Training Video
Contents
      Foreword	i

      Introduction	1

      Water and Light	1

      Culture Water	1

      Photoperiod	2

      Culture Vessels	2

      Water Delivery Systems	2

      Culture Start Up and Maintenance	3

      Starting Cultures	3

      Taxonomy	4

      Collecting Test Organisms	4

      Tank Cleaning	5

      Record Keeping	:	6

      Food Preparation	7

      Citations and Recommended References	8

      Glossary	Glossary-1

      Appendix A:  Apparatus and Equipment List	A-l

      FIGURES

      Figure  1. The General Morphology of Mysids: (A) Lateral View; (B) Dorsal View	1

      Figure  2. Intermittent Flow-Through Water Delivery System	3

      Figure 3. Morphological Characteristics Used in Mysid Identification	4

      Figure 4. Life Cycle of a Mysid	4

      Figure 5. Illustration of Mysid Brood Chamber	5

      Figure 6. Illustration of Mysid Generator	6

      Figure  7.  Data Form for Mysid Cultures  	6

      TABLE

      Table 1. Recommended Culture Conditions for/Amer/camys/s bahia	2

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U'S- ENVIRONMENTAL PROTECTION AGENCY                                                  Culturing Americamysis bahia
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     U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                Culturlng Americamysis bahia
                                                                                   Supplement to Training Video
Introduction
       Americamysis bahia, A. almyra, A. bigelowi, Metamysidopsis ilongata, and Neomysis americana, called
       mysids or opossum shrimp, have all been used in toxicity tests. This guide focuses on Americamysis bahia,
       the EPA-recommended species used in the mysid survival, growth, and fecundity toxicity test (Method
       1007 in EPA, 2002b). Americamysis bahia are found in the coastal waters of the Gulf of Mexico and along
       the Atlantic coast as far north as Rhode Island.

       As shown in 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). Americamysis bahia differ from the other Americamysis
       species by the armature of the telson and the spine-setaes on the thoracic and uropodal endopods
       (Molenock, 1969; Price et al., 1994).
       The culturing procedures
       presented in this supplemen-
       tal guide and illustrated in
       the video were developed to
       meet the specific needs of
       the mysid in each of its life
       stages. This guide and the
       video "Culturing Americamysis
       bahia" (EPA, 2009a) were
       produced by EPA to clarify
       and expand on methods
       explained in the EPA manual
       Methods for Measuring the
       Acute Toxicity of Effluents
       and Receiving Waters to
       Freshwater and Marine
       Organisms, Fifth Edition (EPA,
       2002a). Laboratory person-
       nel who are familiar with the
       culturing and handling pro-
       cedures of the test species
       and the use of healthy test
       organisms are critical for valid
       and successful toxicity test
       results.
Figure 1. The General Morphology of Mysids: (A) Lateral View; (B)
Dorsal View.
 antennule
  antenn'
                                                       dorsal process
                                                              statocyst
                                                              uropod
                                                              telson
                                                              endopod
                                                              exopod
Source: Heard and Price, 2006 as modified from Stuck et al., 1979a.
       The first section of this guide covers the selection and preparation of the water for culturing and presents
       options for water delivery systems. The second section explains how to set up and maintain mysid cultures
       specifically for providing healthy test organisms. The third section provides instructions for collecting young
       of the same age for testing. The fourth section provides details on the food preparation methods used at
       NHEERL-AED in Narragansett, Rhode Island. This guide also includes a glossary and additional references.
       Appendix A provides a list of the apparatus and equipment needed to culture mysids.

Water and Light
       CULTURE WATER
       Culture water is a primary consideration when starting mysid cultures. EPA recommends using natural
       seawater. However, hypersaline brine may be used to make up culture water if natural seawater is not avail-
       able. If natural seawater is used, it must be contaminant-free and filtered through a 0.45 urn screen before
       use to remove particulates and possible predators. The source of the culture water should be uncontami-
                                                                                                     I

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                            Culturing 4m eric am/sis bahia
                                                                               Supplement to Training Video
   nated, consistent, reliable, and periodically checked to ensure the water supports adequate performance
   of the test organisms with respect to survival, growth, and reproduction. More specific instructions for
   the preparation of artificial seawater are listed in EPA's Acute Methods Manual (EPA, 2002a) or can be
   obtained from commercial suppliers. Optimum culture conditions, including water quality, are provided in
   Table 1.

   Table 1. Recommended Culture Conditions for Americamysis bahia
Parameter
Salinity
Temperature
PH
Dissolved oxygen
Ammonia
Nitrite
Nitrate
Alkalinity
Photoperiod
Filtration
Tank Size
Substrate
Biological filter / algal mat
Culture Conditions
25 g/l (20%0 - 30%o)
26C IC
7.8-8.2
7.1 mg/L
O.I-0.3mg/L
<0.05 mg/L
<20 mg/L
ISO mg/L
12-hr light: 12-hr dark to 16-hr
light:8-hr dark
20 urn
10-55 gal
Dolomite, oyster shells, coral
Spirulina subsalsa
   Source: Lussier et a/., 1988.

   Reference toxicant tests should be conducted at least once each month to analyze both the culture water
   being used and to check the mysid mass culture's sensitivity. Recommended reference toxicants are cop-
   per sulfate, cadmium chloride, or sodium dodecyl sulphate.

   PHOTOPERIOD
   For optimum growth and fecundity, the photoperiod for mysid cultures should be 16 hours light and 8 hours
   dark with a light intensity of about 50 - 100 foot-candles. EPA recommends using a system that turns the
   lights on and off gradually so as not to startle the mysids, which can cause them to jump out of the culture
   vessels. Alternatively, the light cycle can be provided using overhead room lights (cool-white fluorescent
   bulbs, approximately 50 ft-c), supplemented with individual grow lights placed over each tank (approximate-
   ly 65 ft-c). This arrangement allows the overhead lights to turn on one hour before the aquaria lights turn
   on and to turn off one hour after they are extinguished.

   CULTURE VESSELS
   Mysids can be cultured in tanks of various sizes. The most commonly used are 20 and 29 gallon aquaria.
   Wider tanks are more suitable for culturingthan taller ones because a large surface area  to volume ratio
   provides both  good oxygen exchange and a larger surface area for these epibenthic organisms that prefer
   to hover over the bottom of the tank. Tanks, as with all culturing equipment, should be cured in the culture
   water for approximately 3-5 days before  being used for organisms.

   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 runoff is discharged
   out of the system (see Figure 2). The flow rate through the culture tanks should be no less than 4-5 liters
   per hour or two complete 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, cop-

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                           Culturing Americomys/s bah/a
                                                                              Supplement to Training Video
                                  Source: EPA 2002b.
       per, brass, or plastic should       Figure 2. Intermittent Flow-Through Water Delivery System
       not be used because they
       could become a source of
       toxicity.

       Recirculating systems also
       can be used to culture mysids
       and should be designed to
       provide the same flow rate
       as the flow-through sys-
       tem. 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.
       This biological filtration system
       serves to oxidize the ammonia and nitrites that can build up in a closed system. A sand filter also may 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" (sudden death of a culture) are less common. Each tank in a static system should be
       supplied with an under  gravel 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.

Culture  Start  Up and Maintenance

       STARTING CULTURES
       Once the culture system and water source are designed, obtained, and seasoned, mysids can be pur-
       chased from a number  of sources. A reliable supplier will certify that the correct species has been shipped.
       Records of the verification  should be retained with a few preserved organisms.  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 card-
       board shipping cartons. The shipping density should be <100 mysids per liter and the container should
       have 2 - 4 cm of 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 so that the
       mysids are not in transit without food for more than 24 hours.

       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 decreas-
       es in temperature and salinity should not exceed 2C or 2%p  - 3%o, respectively, per day.

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

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                               Culturing Americamysis bahia
                                                                                  Supplement to Training Video
       TAXONOMY
       Mysids usually appear trans-
       parent with a yellow, brown,
       or black tint and range from
       4.4 mm to 9.4 mm in length
       (Molenock, 1969). The mor-
       phological characteristics used
       to distinguish A. bahia from
       other mysids are presented in
       Figure 3.

       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 matu-
       rity in about 20 days. Brood
       pouches appear at the age
       of 12 - 16 days and young
       are released at approximately
       20 days. A gravid female is
       identified  by an enlarged and
       darkened brood pouch contain-
       ing 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. Broods are produced
       for several months at a rate of
       one every 4-6 days.
Figure 3. Morphological Characteristics Used in Mysid
Identification
Collecting Test
Organisms
       To conduct toxicity tests using
       mysids, organisms of the
       same age must be collected
       and pooled. To accomplish
       this, gravid females are col-
       lected from the culture tanks
       and their young are  collected
       and held until the proper age
       for starting tests. For testing
Morphological features most useful in identifying Americamysis bahia. a.
male; b. female; c. thoracic leg 2; d. telson; e. right uropod, dorsal; f. male,
dorsal (redrawn from Molenock, 1969; Heard et al, 1987). Note testes in
area where marsupium is located on female and length of male pleopods
as compared to female. Also note the three spines on the endopod of the
uropod (e).

Source: Molenock, 1969; Price et al., 1994
Figure 4. Life Cycle of a Mysid
               Day 20
              First Brood
               Release
     Day 12
  Secondary Sex
  Characteristics

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                            Culturing Americamysis bahia
                                                                              Supplement to Training Video
   needs, assume a reproduc-
   tion rate of two juveniles per
   female per day because not all
   females will release their young
   on the same day. Collect the
   gravid females from a minimum
   of three culture tanks. While
   identifying and selecting gravid
   females for the brood chamber,
   the sex ratio and density of each
   tank should be determined and
   adjusted, if needed, to maintain
   a ratio of 2 females:! male.
   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 L Nalgene
   beaker that is placed inside a
   separatory funnel 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.
Figure 5. Illustration of Mysid Brood Chamber
                               INFLOW
                                            OUTFLOW
                                             NETTED
                                            CHAMBER
                                      SEPARATORY
                                         FUNNEL
                                          NETTED
                                        CULTURE DISH
                                  _-^
Source: Lussier, et a/., 1987.
  Once the females are placed in the brood chamber, provide food and gentle aeration by either placing
  an airstone in the neck of the separatory funnel or 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 or stop the flow of water and slowly drain the separatory cham-
  ber into a 300 urn mesh cup placed in a culture dish. To prevent injury to the young mysids, partially
  submerge the mesh cup in culture water within the culture dish before draining the brood chamber. While
  the water is draining, gently lift and dunk the beaker 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. 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 urn 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 urn 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 juve-
  niles' 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, which will
  clear the dolomite filter.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                             Culturing Americamysis bahia
                                                                                Supplement to Training Video
                                                                             .  - ^ ..t.  , 
   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 in the previous sec-
   tion "Waterand Light, Culture
   Vessels," before using them in
   culture tanks.

   RECORD KEEPING
   Culture tanks should be
   monitored and all conditions
   recorded on data forms that
   are kept in a permanent file.
   These forms are used to
   assess any problems that may
   occur with the cultures and
   assist in eliminating possible
   causes. The forms also serve
   as a record for testing labora-
   tories to verify that their test
   organisms were raised using
   proper culture techniques.
    Figure 6. Illustration of Mysid Generator

                       Pump Return
                       Tube
Filter
  Incoming
  Siphon
        Culture Tank
        (75 L)
Overflow
       Collection
       Tank
    Source: Lussier et a/., 1988.
   Figure 7 is a data sheet adapted from the one used by AED-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 measurement of temperature, pH, salinity, and dissolved
   oxygen; seawater and airflow checks; and feeding (twice daily).
Figure 7. Data Form for Mysid Cultures
Dote







Temp
C







pH
SU







Salinity
%0







DO
mg/L







SW
Flow







Air
Flow







Mysids.
Fed







Comments








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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                Culturing Americamysis bahia
                                                                                  Supplement to Training Video
Food Preparation
       Mysid cultures are fed Artemia nauplii (newly-hatched brine shrimp) twice each day at a rate that ensures
       live Artemia are always available in the tanks (approximately 150 Artemia nauplii per mysid per day). The
       Artemia should be cultured in the laboratory in order to provide 24 - 48 hour old nauplii on a daily basis.
       Artemia cysts are available from commercial suppliers. Each shipment of Artemia received should be
       analyzed for priority pollutants and should be tested on a small batch  of mysids to ensure that good mysid
       growth and reproduction occur before the Artemia are fed to entire mysid cultures. Food supplements are
       commercially available and are used more often when using artificial seawater for culturing.

       Culture the Artemia-by adding dry cysts to clean seawater at a rate of  approximately 10 ml cysts to 1 L
       seawater (ASTM, 1998). A separatory funnel works well for culturing Artemia. Inverted two-liter plastic
       bottles also have been used by cutting out their bottoms and inserting a rubber stopper with a flexible tube
       and pinch clamp.

       After placing the water and cysts into the culture chamber, aerate vigorously to keep the cysts (and eventu-
       ally the newly-hatched nauplii) in suspension. Deliver the filtered air through a 1 ml pipet by resting the-tip
       of the pipet at the bottom of the neck  of the chamber. This keeps the nauplii from settling and depleting
       the oxygen supply.
     IMPORTANT NOTE:
     The nauplii must be aerated
     if they remain unused for
     more than a few minutes.
     Without aeration the nauplii
     will begin to die.
The cysts will hatch in approximately 24 hours. Newly-hatched Artemia
nauplii are more nutritious than older ones and are the appropriate
size for feeding early juvenile mysids. To harvest the nauplii for feeding,
remove the air supply and allow the cysts and nauplii to separate for five
minutes. The empty cysts will float and the nauplii will descend to the
neck of the chamber. The nauplii are attracted to light, so a light source
placed at the bottom of the chamber and/or a dark cover or hood
placed on the top will hasten the separation process.
       Drain the nauplii through the stop clamp or siphon them from the bottom of the chambers. If the nauplii
       are drained through the stop cock, the first plug of unhatched cysts that collect at the neck of the cham-
       ber should be discarded and not mixed with the nauplii. Drain only the hatched nauplii (the bright orange
       suspension),  leaving behind the empty cysts. The nauplii should be drained through a 150 urn screen and
       rinsed with clean seawater to remove any chemicals released during hatching.

       To determine the correct amount of Artemia for feeding, an aliquot of the hatched Artemia 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 and that mysids are being fed a consistent
       amount of food.

       Once the Artemia 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 Artemia per mysid per day, or 75
       Artemia per mysid per feeding. Feeding the mysids in two feedings, 8-12 hours apart ensures there are
       always live Artemia available for the mysids.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                              Culturing Americamysis bahia
                                                                                 Supplement to Training Video
Citations  and Recommended References

       American Society for Testing and Materials. 1998. Standard Practice for Using Brine Shrimp Nauplii as
               Food for Testing Animals in Aquatic Toxicology. ASTM Designation E-1203-98. Philadelphia, PA.

       Blaxter, 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.

       EPA. 1991. Technical Support Document for Water Quality-based Toxics Control. U.S. EPA Office of Water
               Enforcement and Permits, Washington, D.C. EPA-505-2-90-001.

       EPA. 2000a. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR
               Part 136). Office of Water, Washington, D.C. EPA 821-B-00-004.

       EPA. 2000b. Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications
               Under the National Pollutant Discharge Elimination System Program.  Office of Wastewater
               Management, Washington, D.C. EPA 833-R-00-003.

       EPA. 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
               and Marine Organisms, Fifth Edition. (Acute Methods Manual). Office of Water, Cincinnati, OH.
               EPA-821-R-02-012.

       EPA. 2002b. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters
               to Marine and Estuarine Organisms, Third Edition. (Chronic Methods Manual). Office of Water,
               Cincinnati, OH. EPA-821-R-02-014.

       EPA. 2009a. Culturing Americamysis bahia. Supplement to Training Video. Whole Effluent Toxicity Training
               Video Series, Saltwater Series. March 2009. EPA 833-C-09-001.

       EPA. 2009b. Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests. Supplement to
               Training Video. Whole Effluent Toxicity Training Video Series, Saltwater Series. March 2009. EPA
               833-C-09-001.

       Farrell, D.H. 1979. Guide to the shallow-water mysids from Florida. Fla. Dept.  Environ. Reg., Techn. Ser.
       Fotheringham, 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.

       Heard, R.W., W.W. Price, and K.C. Stuck. 1987. Mysid Identification Workshop. The Gulf Coast Research
               Laboratory, Ocean Springs, Mississippi (unpublished.)

       Heard, R.W. and W.W. Price. 2006. A taxonomic Guide to the Mysids of the South Atlantic Bight. U.S.
               Department of Commerce, National Oceanic and Atmospheric Administration.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                            Culturing Americamysis bahia
                                                                               Supplement to Training Video
   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, and J. Sewall. 1987. Guidance manual for conducting 7-day mysid survival/
           growth/reproduction study using the estuarine mysid, Mysidopsis bahia. Contribution No. X106.
           In: Schimmel, S.C., ed. Users guide to the conduct and interpretation of complex effluent toxic-
           ity tests at estuarine/marine sites. Environmental Research Laboratory, U.S.  Environmental
           Protection Agency, Narragansett, Rhode Island. Contribution No. 796, 265 pp.

   Lussier, S.M., A. Kuhn and R. Comeleo. 1999. An evaluation of the seven-day toxicity test with
           /Amer/camys/s bahia (formerly Mysidopsis bahia). Environ. Toxicol. and Chem. 18:2888-2893.
           [Errata: in the section on Experimental Design, the test chamber should read "200-ml plastic cup"
           not "30-ml."]

   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. Zool. Bot. 15(3):113-116.

   Nimmo, D.R. and T.L. Hamaker. 1982.  Mysids in toxicity 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 par-
           ticulates 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.C. pp. 41-50.

   Nimmo, D.R., T.L. Hamaker, C.A. Sommers. 1978. Culturing the mysid (Mysidopsis bahia) 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., R.W. Heard and L. Stuck. 1994. Observations on the genus Mysidopsis sars. 1864 with the
           designation of a new genus, Americamysis, and the descriptions of Americamysis aliens and A.
           stuck! (Peracarida: Mysidacea: Mysidae), from the Gulf of Mexico. Proc. Biol. Soc. Wash. 107: 680-
           698.

   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 Mysiacea from coastal
           and shelf water of the Eastern Gulf of Mexico. Gulf Res. Rept.  6(3):239-248.

   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.

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                                                                                 Culturing Americamysis bahia
                                                                                    Supplement to Training Video
    U.S. ENVIRONMENTAL PROTECTION AGENCY
       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.

       EPA references are available online atwww.epa.gov/npdes.

       If you need additional copies of this document, you can download it at:
               www.epa.gov/npdes/wqbasedpermitting.
10

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                 Culturing Americomysis bahia
                                                                                    Supplement to Training Video
Glossary
       Artemia. The marine invertebrate (referred to as brine shrimp) used as the recommended food source for
               mysid cultures and test organisms; Brazilian or Colombian strains are preferred because the sup-
               plies are found to have low concentrations of chemical residues and nauplii are of suitably small
               size.

       Crash. Sudden (overnight) death of cultured organisms in a tank.

       Cyst. The life stage of unhatched Artemia.

       Epibenthic. Pertaining to the area just above the sediment.

       Fecundity. Productivity or fertility as measured in the mysid test as the percentage of females with eggs
               in the oviduct and/or brood pouch.

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

       Mysid (Americamysis bahia). An estuarine crustacean, formerly known as Mysidopsis bahia, ranging 4.4
               mm to 9.4 mm in length found from the Gulf of Mexico and along the Atlantic  coast as far north as
               Rhode Island; used in test procedures as an indicator species for aquatic toxicity.

       Nauplii. Free-swimming microscopic larvae stage characteristic of copepods, ostracods, barnacles, etc.
               typically with only three pairs of appendages.

       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.

       WET (Whole effulent toxicty). The total toxic effect of an effluent measured directly with a toxicty text.
                                                                                             Glossary-1

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ISC) U'S- ENVIRONMENTAI- PROTECTION AGENCY                                                  Culturing Americamysif bahia
V=^                                                                                              Supplement to Training Video
                                                  Intentionally Left Blank
Glossary-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                             Culturing Americamysis bahia
                                                                                Supplement to Training Video
Appendix A
Apparatus and Equipment List
       Air line and air stones. For aerating cultures, brood chambers, and holding tanks, and supplying air to test
              solutions with low DO.

       Air pump. For oil-free air supply.

       Balance. Analytical, capable of accurately weighing to 0.00001 g.

       Beakers or flasks. Six, borosilicate glass or non-toxic plasticware, 2 - 3 L for making test solutions.

       Brine shrimp (Artemia) culture unit. See "Food Preparation" section.

       Depression glass slides or depression spot plates. Two for observing organisms.

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

       Droppers, and glass tubing with fire polished edges. 4 mm inner diameter (ID), for transferring
              organisms.

       Environmental chamber or equivalent facility with temperature control (26  1C).

       Facilities for holding and acclimating test organisms.

       Light box. For illuminating organisms during examination.

       Meters: pH and DO, and specific conductivity. For routine physical and chemical measurements.

       Mysid (Americamysis bahia) culture unit. See "Culture Start Up and Maintenance" section. The test
              requires a minimum of 240 7-day old (juvenile) mysids.

       NITEX or stainless steel mesh sieves. 150 urn and 100 Mm for concentrating organisms; 1 mm mesh
              and  300 |jrn mesh for collection of juveniles.

       Pipet bulbs and fillers. Propipet, or equivalent.

       Pipets, automatic. Adjustable,  1 - 100 ml_.

       Pipets, serological. 1-10 mL, graduated.

       Pipets, volumetric, Class A. 100 ml.

       Reference weights, Class S. For checking performance of balance.

       Refractometer or other method. For determining salinity.

       Separatory funnels, 2-liters. Two to four for culturing/Artem/a.

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

       Thermometers, bulb-thermograph or electronic-chart type. For continuously recording temperature.

       Thermometers, glass or electronic, laboratory grade. For measuring water temperatures.

                                                                                               A-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                                  Culturing Amencamysis bahia
                                                                                    Supplement to Training Video
        Thermometers. National Bureau of Standards Certified (see EPA, 2002b). Used to calibrate laboratory
               thermometers.

        Volumetric flasks and graduated cylinders. Class A, borosilicate glass or non-toxic plastic labware,
               50 - 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.

        Water purification system. Millipore Milli-Q deionized water or equivalent.
A-2

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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 /HOLE bFFLUENT IOXICITY  I RAINING VIDEO SERIES  Saltwater Series
         Mysid (Americamysi's bah/a)
         Survival, Growth, and  Fecundity
         Toxicity Tests
         Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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                      NOTICE

 The revision of this guide has been funded wholly or in part by the
  Environmental Protection Agency under Contract EP-C-05-063.
Mention of trade names or commercial products does not constitute
           endorsement or recommendation for use.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Xtmericam/sis bah/a) Survival, Growth, and Fecundity Toxicity Tests
                                                                                   Supplement to Training Video
Foreword
       This guide serves as a supplement to the video "Mysid (Americamysis bahia) Survival, Growth, and
       Fecundity Toxicity Tests" (EPA, 2009a). The methods illustrated in the video and described in this sup-
       plemental guide support the methods published in the U.S. Environmental Protection Agency's (EPA's)
       Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and
       Estuarine Organisms, Third Edition (EPA, 2002a), referred to as the Saltwater Chronic Methods Manual.
       The video and this guide provide details on preparing for and conducting the test based on the expertise of
       personnel at the following EPA Office of Research and Development (ORD) laboratories:

           National Health and Environmental Effects Research Laboratory (NHEERL) - Atlantic Ecology Division
           in Narragansett, Rhode Island

           NHEERL - Gulf Ecology Division in Gulf Breeze, Florida

           National Exposure Research Lab (NERL) - Ecological Exposure Research Division (EERD) in
           Cincinnati, Ohio

       This guide and its accompanying video are part of a series of training videos produced by EPA's Office of
       Wastewater Management. This Saltwater Series includes the following videos and guides:

           "Mysid (/Amer/camys/s bahia) Survival, Growth, and Fecundity Toxicity Tests"

           "CulturingX\mer/camys/s bahia"

           "Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia punctulata"

           "Red Algal (Champia parvula) Sexual  Reproduction Toxicity Tests"

           "Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
               and Growth Toxicity Tests"

       The Freshwater Series, released in 2006,  includes the following videos and supplemental guides:

           "Ceriodaphnia Survival and Reproduction Toxicity Tests"

           "Culturingof Fathead Minnows (Pimephales promelas)"

           "Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests"

       All of these videos are available through the National Service Center for Environmental Publications
       (NSCEP) at 800 490-9198 or nscep@bps-lmit.com.

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U.S. ENVIRONMENTAL PROTECTION AGENCY         Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                              Supplement to Training Video
                                             Intentionally Left Blank

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    U.S. ENVIRONMENTAL PROTECTION AGENCY       Mysid (Americomysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                             Supplement to Training Video
Contents
      Foreword	i
      Tables	iv
      Figures	iv
      Introduction	1
      Background	1
      Maintaining and Feeding Cultures	1
      Culture Maintenance	1
      Feeding	2
      Collecting Juveniles for Test Use	2
      Conducting the Test	.3
      Effluent Sampling	3
      Dilution Preparation	3
      Routine Chemistries	4
      Test Chambers	5
      Test Organisms	6
      Feeding	6
      Renewals	6
      Terminating the Test	8
      Test Acceptability and Data Review	11
      Citations and Recommended References	12
      Glossary	Glossary-1
      Appendix A: Summary of Test Conditions and Test Acceptability Criteria	A-l
      Appendix B: Apparatus and Equipment List	B-l
      Appendix C: Reagents and Consumable Materials	C-l
      Appendix D: Preparing Hypersaline  Brine (MSB)	D-l

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U.S. ENVIRONMENTAL PROTECTION AGENCY       Mysid (Americomysis bohia) Survival, Growth, and Fecundity Toxicity Tests
                                                                           Supplement to Training Video
   FIGURES
   Figure 1. The General Morphology of Mysids. (A) Lateral View; (B) Dorsal View	2
   Figure 2. Apparatus for Collection of Juvenile Mysids from Gravid Females  	2
   Figure 3. Data Form for the Mysid Survival and Fecundity Toxicity Test - Water Quality Data	5
   Figure 4. Data Form for the Mysid Survival and Fecundity Toxicity Test - Survival and
   Fecundity Data	7
   Figure 5. Data Form for the Mysid Survival and Fecundity Toxicity Test - Dry Weight Measures .. 8
   Figure 6. Mature Female A. bah/a with Eggs in Oviducts. Lateral view (top) Dorsal view (bottom) 9
   Figure 7. Mature Female A. bahia with Eggs in Oviducts and Developing Embryos in Brood Sac.
   Lateral view (top) Dorsal view (bottom)	10
   Figure 8. Mature Male A. bahia. Lateral view (top) Dorsal View (bottom)  ..."	10
   Figure 9. Immature A. bahia. Lateral view (top) Dorsal view (bottom)	11

   TABLES
   Table 1. Monitoring Schedule	4
   Table A-l. Summary of Test Conditions and Test Acceptability Criteria for Americamysis bahia
   7-day Survival, Growth, and Fecundity Toxicity Test	A-l
   Table D-l. Preparation of Test Solutions at a Salinity of 20%o Using HSB for a Final Test
   Concentration Volume of 2000 mL	D-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americomysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                  Supplement to Training Video
Introduction

       This supplemental guide accompanies the Environmental Protection Agency's (EPA's) video to provide
       instructions for conducting the standard 7-day survival, growth, and fecundity toxicity test using the
       mysid, Americamysis bahia (EPA, 2009a; EPA, 2009b). The test method is found in Short-term Methods
       for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms,
       Third Edition (EPA, 2002a). The methods presented in this guide and the video are based on the expe-
       rience and standardized practices developed at EPA's Office of Research and Development's (ORD's)
       National Health and Environmental Effects Research  Laboratory-Atlantic Ecology Division (NHEERL-AED)
       in Narragansett, Rhode Island. The material  presented in both the video and this guide summarizes the
       methods but does not replace a thorough review and understanding of the methods by laboratory person-
       nel before conducting the test.
Background
       Under the National Pollutant Discharge Elimination System (NPDES) program (Section 402 of the Clean
       Water Act), EPA uses toxicity tests to monitor and evaluate effluents for their toxicity to biota and their
       impact on receiving waters. By determining acceptable or safe concentrations for toxicants discharged
       into receiving waters, EPA can establish NPDES permit limitations for toxicity. These whole effluent toxicity
       (WET) permit limitations regulate pollutant discharges on a whole effluent effect basis rather than solely by
       a chemical-specific approach.

       The mysid survival, growth, and fecundity toxicity test (Test Method 1007.0 in EPA, 2002a) is used by EPA
       for determining the toxicity of marine or estuarine discharges by measuring specified endpoints after a
       7-day exposure period. Whole effluent toxicity methods measure the synergistic, antagonistic, and addi-
       tive effects of all the chemical, physical, and additive components of an effluent that adversely affect the
       physiological and biochemical functions of the test organisms. Therefore, healthy organisms and correct
       laboratory procedures are essential for valid test results. Laboratory personnel  should be very familiar with
       the test methods and with mysid handling techniques before conducting a test.

       This supplemental guide covers the procedures for conducting the test according to EPA's promulgated
       methods (40 CFR Part 136; EPA, 2002c) and also provides some helpful information that is not presented
       in the Saltwater Chronic Methods Manual (EPA, 2002-a).

Maintaining  and  Feeding  Cultures

       CULTURE MAINTENANCE
       /4mericamys/s bahia (mysids, or opossum shrimp) are estuarine invertebrates generally found in the coastal
       waters of the Gulf of Mexico and along the Atlantic coast as far north as Rhode  Island (see Figure 1). They
       usually appear transparent with a yellow, brown, or black tint and range from 4.4 mm to 9.4 mm in length
       (Molenock, 1969). Adult mysids can be collected from the field, however, they must be verified taxonomi-
       cally as the correct species before being placed in cultures for test use (Price et al., 1994). Alternatively,
       commercial suppliers provide adults for cultures and juveniles for cultures or testing. The supplier should
       verify that the correct species is sent.

       Cultures should be maintained in glass aquaria supplied with flow-through or recirculating seawater
       (Lussier et al., 1988). The water temperature should be 26C and salinity between 20%o to 30%0 and
       should not fluctuate more than 2C or 2%o per day, respectively. The light regime recommended for cultur-
       ing is 16 hours light and 8 hours dark. The light should be phased on and off gradually so as not to startle
       the mysids.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                             Mysid (Americamyfis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                 Supplement to Training Video
       FEEDING
       Mysids are fed <24-hr old
       Artemia nauplii (newly hatched
       brine shrimp) twice daily.
       Feeding amounts should be
       adequate to provide live food
       at all times for the mysids
       to feed upon. Approximately
       150 Artemia per mysid per
       day is recommended. Artemia
       supplies should be checked
       periodically for contamination
       and hatch rates.

       Detailed instructions on
       culturing/4rtem/a are pre-
       sented in  the video "Culturing
       Americamysis bahia," and its
       accompanying supplemental
       guide, and in the EPA manual
       Methods for Measuring the
       Acute Toxicity of Effluents
       and Receiving Waters to
       Freshwater and Marine
       Organisms, Fifth Edition
       (EPA, 2009b; EPA, 2002b).

Collecting Juveniles

for Test Use

       The 7-day survival, growth, and
       fecundity  toxicity test must be
       started with 7-day old mysids that
       are all within 24 hours age of each
       other. Seven-day old juveniles are
       needed in sufficient number to
       randomly  select five juveniles for
       each replicate. For a test with five
       effluent concentrations and one
       control, with 8 replicates at each
       concentration, it is recommended
       to have approximately 240 -
       300, 7-day old mysids available
       to choose from. Avoid using any
       mysids that appear injured.

       To  collect juveniles and to be
       assured of their age range (within
       24-hours  age), a brood chamber
       is used (see Figure 2). The brood
       chamber  is set up eight days
       before the start of the test.
Figure 1. The General Morphology of Mysids. (A) Lateral View; (B)
Dorsal View
antennule
antenna
                                               dorsal process
                                                         statocyst
                      thoracic segments    dorsal process
 Source: Heard and Price, 2006 as modified from Stuck et a/., 1979.
Figure 2. Apparatus for Collection of Juvenile Mysids from Gravid
Females
                             INFLOW
                                          OUTFLOW
                                           NETTED
                                          CHAMBER
                                    SEPARATORY
                                       FUNNEL
                                        NETTED
                                       'CHAMBER
                                      CULTURE DISH
Source: Lussier et a/., 1987.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                              Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                   Supplement to Training Video
       Gravid females selected from a minimum of three culture tanks are placed in a netted chamber inside a
       funnel. Gravid females are those ready to release their young and are identified by dark spots in their brood
       pouches. Because not all of the females will release young on the same day, an estimate of two juveniles
       per female per day should be used to determine the number of gravid females needed. Therefore, to have
       sufficient mysids for test initiation, approximately 125 - 150 gravid females should be placed in the brood
       chamber.

       Twenty-four hours after placing the females in the brood chamber, or seven days before the test start date,
       remove the netted chamber containing the gravid females from the brood chamber allowing the juveniles to
       escape through the screened bottom. Return the females to the culture tanks and drain the juveniles from
       the funnel into a mesh cup placed in a dish containing culture water. To prevent injury to the test animals,
       gently rinse the sides of the funnel as it drains. These juveniles, all born within the last 24 hours should
       be counted and transferred into a separate tank where they will be held for the next seven days. Because
       stocking density is very important to the rate of juvenile development, no more than 300 juveniles should
       be held in a 10-gallon tank. If the holding tank used is a static system,  half of the water must be replaced
       every other day with new culture water.

       Nutrition and temperature are important factors in mysid development (Lussier et al., 1999). During the
       7-day holding period maintain the holding tanks at 26C - 27C with a  salinity similar to the culture/test
       water. If necessary, the salinity should be gradually adjusted (<2%o/day) to the desired test salinity (20%o -
       30%o) during this holding period. Feed the juveniles <24-hour old Artemia nauplii twice daily.
Conducting the Test
  Under the NPDES program, lapsed
  time from sample collection to first
  use of that sample in a toxicity
  test (i.e., test initiation) must not
  exceed 36 hours.  If stored correctly,
  the sample may be used for test
  renewals at 24 hours, 48 hours,
  and/or  72 hours after test initiation.
EFFLUENT SAMPLING
Effluent sampling should be conducted according to the EPA Saltwater
Chronic Methods Manual (EPA, 2002a) and any conditions specified
in a regulatory permit. In static renewal tests, each grab or composite
sample may be used to prepare test solutions for renewal at 24, 48,
and/or 72 hours after first use if stored between  0C - 6C, with mini-
mum head space. According to the EPA 2002 promulgated methods, for
WET samples with a specified storage temperature of 4C, storage at a
temperature above the freezing point of water to  6C shall be acceptable
(0C -  6C).  EPA has further clarified that hand-delivered samples used
on the  day of collection do not need to be cooled to 0C - 6C prior to
test initiation (EPA, 2002c).
  Dilution Water
  The type of dilution water used to make the test concentrations is
  dependent on the objectives of the test. Any specific requirements
  included in NPDES permits should be followed.  The Saltwater Chronic
  Methods Manual (Section 7) provides the following guidelines:
    If the test is conducted to estimate the absolute chronic toxicity
  of the effluent, synthetic dilution water should be used. If the cultures
  were maintained in different water than used for dilution water, a
  second set of control replicates should be conducted using the culture
  water.
    If the test is conducted to estimate the chronic toxicity of the
  effluent in uncontaminated receiving waters, the test (com.)
                                DILUTION PREPARATION
                                To start a test, warm the effluent to
                                26C  1C slowly to avoid exceed-
                                ing the desired temperature. This
                                is accomplished using a water bath
                                and monitoring the temperature
                                closely. A temperature of 26C  1C
                                should be maintained throughout the
                                7-day test period and the instanta-
                                neous temperature must not deviate
                                by more than 3C during the test.

                                Once the effluent and the dilution
                                water reach the desired tempera-
                                ture, the dilutions are prepared.

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                              Mysid (Americomysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                    Supplement to Training Video
Dilution Water (cont.)
can be conducted using a grab sample of the receiving waters collected
outside the influence of the outfall, other uncontaminated waters, or
standard dilution water with the same salinity as the receiving waters.
If the cultures were maintained in different water than used for dilution
water, a second set of control  replicates  should be conducted using the
culture water.
  If the  test is conducted to estimate the additive or mitigating
effects of the effluent on  already contaminated receiving
waters,  the test must be conducted using receiving waters collected
outside the influence of the outfall. Controls should be conducted using
both receiving  water and culture water.
     tions because less dilution is needed to adjust to the proper salinity.
Because the marine/estuarine
species used for testing are salin-
ity sensitive, the effluent must be
adjusted to the proper salinity before
preparing the test concentrations.
Hypersaline brine is recommended
for adjusting the effluent salinity.
Appendix D provides instructions for
preparing the brine solution (EPA,
2002a). To prepare test concentra-
tions at the desired salinity, adjust
the diluent (deionized water) with the
hypersaline brine before adding it to
the effluent. Using hypersaline brine
instead of seawater allows the test to
be run at higher effluent concentra-
     Use a minimum of five exposure concentrations and a control with a minimum of eight replicates per
     concentration. The Saltwater Chronic Methods Manual recommends the use of a 0.5 dilution factor, which
     provides precision of + 100%. Test precision shows little improvement as the dilution factor is increased
     beyond 0.5, and declines rapidly if a smaller dilution factor is used. Approximately 3 L of test solution are
     needed each day for a test conducted with 8 replicates of 5 concentrations and a control.

     ROUTINE CHEMISTRIES
                Once the various concentrations are prepared, set aside one aliquot of each for conducting
                routine chemistries. By setting these aside, the chemistries can be performed without con-
                 taminating the actual test solutions with the probe. For test initiation and renewals, measure
                  and record the dissolved oxygen (DO) at the  beginning and end of each 24-hour renewal in
                   at  least one test  chamber of each test concentration and in the control. If the DO falls below
                  4.0 mg/L in any replicate, aerate all concentrations and the control. Take care not to cause
                 excess turbulence that can cause physical stress to the organisms.

     Dissolved oxygen, temperature,  pH, and salinity must be measured on each new sample. Dissolved oxygen
     is measured at the beginning and end of each 24-hour renewal in at least one test chamber of each test
     concentration and in the control. Measuring salinity at the beginning and end of each 24-hour renewal is pre-
     ferred but not required. The salinity, temperature, and  pH of the effluent sample must be measured at the end
     of each 24-hour exposure period in one test chamber at each concentration and in the control. See Table 1.
     Table 1. Monitoring Schedule
Parameter
Dissolved Oxygen1 2
Temperature13
pH'3
Salinity1'2
Monitoring Frequency
Each New Sample
X
X
X
X
24-hr Exposure Period
Beginning
X


X
End
X
X
X
X
      / Measured in each new sample (100% effluent or receiving water) and in control.
      2 Beginning and end measurement on one replicate in each concentration and the control.
      3 End measurement on one replicate in each concentration and the control.

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 U.S. ENVIRONMENTAL PROTECTION AGENCY
                                           Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                 Supplement to Training Video
   These parameters should fall within the recommended ranges for conducting the test and they should be
   recorded on the test data sheet. The recommended test conditions are presented in Appendix A and a
   sample water quality data sheet is provided in Figure 3.
Figure 3. Data Form for the Mysid Survival and Fecundity Toxicity Test - Water Quality Data
   Test:	
   Start Date:.
Salinity:.

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7



Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day?

TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP

TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
TEMP















TEMP














Salinity















Salinity














DO















DO














pH















pH














TRTMT















TRTMT














TEMP















TEMP














Salinity















Salinity














DO















DO














pH















pH














Source: EPA, 2002a.

   TEST CHAMBERS
   The test chambers should be readied before the effluent concentrations are prepared. EPA recommends
   using 8 oz disposable plastic drinking cups or 400 ml glass beakers to conduct this test. The test cham-
   bers are presoaked in clean seawater and labeled with colored tape. Each concentration is indicated by a
   different color tape with the replicate number (1 - 8) written on it. The use of different colored tape makes
   renewals easier because  all of the replicates of one concentration can be identified quickly.

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U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis boh/a) Survival, Growth, and Fecundity Toxicity Tests
                                                                               Supplement to Training Video
                                                                               >  

  Once the cups are prepared and the effluent solutions have been adjusted to within the proper parameter
  ranges, each test solution is distributed to eight replicate cups. Each replicate should contain approximate-
  ly 150 ml. The cups are placed in holding trays that are randomly placed in a temperature-controlled water
  bath. The holding trays should be labeled with the same colored tape and replicate numbers as the cups
  which allows for easier collection and replacement of the  randomized cups during renewals. The cups will
  stay in the same randomized positions for the duration of the test. Specific directions for test randomiza-
  tion are provided in Appendix A of the Saltwater Chronic Methods Manual (EPA, 2002a).

  TEST ORGANISMS
  Juvenile mysids should be collected from gravid females obtained from at least three separate culturing
  tanks. To begin a test with five effluent concentrations and a control, each with eight replicates, a mini-
  mum of 240 juveniles  are needed. Having more than 240 juveniles allows for extra juveniles from which to
  choose. Select juveniles at random, but avoid  using any that appear injured.

  Juvenile mysids are assigned to the test chambers at a density of five mysids per chamber.  The juveniles
  are randomly selected from the 7-day old juvenile pool and pipetted using a large bore (4 mm inner diam-
  eter [ID]) pipet into small presoaked ampules, two to three at a time. The open covers of the ampules
  serve as handles. This random selection and assignment is continued until all of the ampules contain five
  mysids. As the mysids are placed in these ampules, a minimum amount of water should be  transferred with
  them so that the effluent concentrations are not diluted.

  To transfer the mysids to the test chambers, the ampules should be dipped below the water level in each
  cup and gently rinsed to deposit the mysids. Pouring the mysids from above the water surface may cause
  injury. The test chambers should remain in the water bath while this transfer is made.

  FEEDING
  Once the test has been set-up, the mysids are fed.  The initial feeding rate is 0.5 ml of a food solution made
  from 4.0 mL concentrated Artemia nauplii in 80 ml of uncontaminated, filtered seawater. This concentra-
  tion of nauplii should yield a level of approximately  150 24-hr old nauplii per mysid per day.  This amount
  of food solution should provide the test organisms  with a  sufficient number of live Artemia for the next 24
  hours until test renewal. Immediately after renewal each day, feed the mysids 0.25 ml of food solution.
  Another 0.25 ml should be fed 8-12 hours later.  The food should be dispensed using an automatic pipet
  and the food solution should be swirled before pipetting to ensure an even distribution of the Artemia. After
  feeding the mysids, cover the test chambers to prevent evaporation or contamination.

  RENEWALS
  To conduct the daily renewals, collect the test cups from the water bath starting with the control and
  working toward the higher concentrations. Measure and record the temperature, salinity,  DO, and pH in a
  composite aliquot of a minimum of two randomly selected replicates from each concentration (see Figure
  3). If the DO concentration falls below 4 mg/L in any one of the exposure chambers, all chambers must be
  gently aerated at a rate of approximately 100  bubbles/minute. During renewals the mysids  in each cham-
  ber should be counted and the survival recorded on the test data sheets. Any dead animals should be
  discarded. A sample survival and fecundity data sheet is presented as Figure 4.

  To renew the effluent,  pour or siphon off the old effluent solution  into a white tray or a large beaker placed
  on a light table. Either of these receptacles will clearly show any mysids that are accidentally removed.
  Slowly pouring the effluent from the cups works well because mysids tend to swim against the current and will
  swim towards the back of the cups. If a mysid is poured out with the old effluent it should be pipetted back into
  the exposure chamber and recorded as "returned during renewal" on the test data sheet. When removing
  the old effluent, a pipet should be used to clean any uneaten Artemia from the bottom of the chamber.

  To add the new effluent solution to the chamber, gently pour approximately 150 ml of the appropriate solu-
  tion down the side of the chamber avoiding as much turbulence as possible. This renewal procedure must

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 U.S. ENVIRONMENTAL PROTECTION AGENCY
                                            Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                   Supplement to Training Video
    be repeated on days two through six of the exposure period. All data should be carefully recorded on the
    data sheets each day.

    Immediately after renewal each day, feed the mysids 0.25 ml of food solution. Another 0.25 mL should
    be fed 8-12 hours later. If the survival rate in any replicate drops below 50%, the food provided to that
    replicate should be reduced by half. Detailed instructions for culturing Artemia are provided in the video
    "Culturing/4mericamys/s bahia" and in its supplemental guide (EPA, 2009b).
Figure 4. Data Form for the Mysid Survival and Fecundity Toxicity Test - Survival and Fecundity Data
    Test:	
    Start Date:
Salinity:.
Treatment/
Replicate
\
2
3
Control 4
5
6
7
8
1
2
3
1 4
5
6
7
8
1
2
3
2 4
5
6
7
8
	 1
Day 1
Alive

























Day 2
Alive

























Day 3
Alive

























Day 4
Alive

























Day5
Alive

























Day 6
Alive

























Day 7
Alive

























Females
vfleggs

























Females
No eggs

























Males

























Imma-
ture!

























Source: EPA, 2002a.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                             Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                  Supplement to Training Video
Terminating the Test
       On the last day of the 7-day exposure, the replicates are checked for survival and fecundity and the animals
       are prepared for growth measurements. The mysids are not fed on the last day of the test so that total
       weights do not reflect the added weight of any undigested Artemia.

       In preparation for the test termination, prepare small pieces (1 cm2) of clean, light-weight aluminum foil by
       labeling them with sequential numbers. Gloves should be worn or forceps should be used to handle the
       aluminum because oils from skin could affect weight differences. After they are numbered, these pieces
       of foil should all be dried, tared, and their weights recorded on the growth-data sheet. The sample growth-
       data sheet is presented as Figure 5.
    Figure 5. Data Form for the Mysid Survival and Fecundity Toxicity Test - Dry Weight Measures
       Test:	
       Start Date:
Salinity:.
Treatment/
Replicate
1
2
3
Control 4
5
6
7
8
1
2
3
1 4
5
6
7
8
1
2
3
2 4
5
6
7
8
	 L_
Pan#

























Tare Wt.

























Total Wt.

























Organism Wt.

























# of Organisms

























Wt./Organism

























    Source: EPA, 2002a.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                          Mysid (Americamysis bah/a) Survival, Growth, and Fecundity Toxicity Tests
                                                                               Supplement to Training Video
   After the aluminum is prepared,
   pick up the test chambers
   in the same manner as for
   conducting a renewal. That
   is, collect all of the replicates antennuie
   of one concentration at
   one time, starting with the
   control. Final water quality
   measurements, including DO,
   temperature, salinity, and pH
   should be measured on ali-
   quots taken from several test
   chambers in each concen-
   tration and the control and
   recorded  (see Figure 3).
   First, remove dead mysids
   from the test chambers and
   record the final survival count
   for each replicate on the test
   data sheet (see Figure 4).
   The minimum requirement
   for an acceptable test is 80%
   survival in the controls.
Figure 6. Mature Female A. bahia with Eggs in Oviducts. Lateral
view (top) Dorsal view (bottom)
                 eyestalk
                           carapace
                                                       statocyst

                                                            telson
                                                              .telson
                                 developing brood sac
                               oviducts with developing ova
uropod
                                   Source: Lussier, Kuhn, and Sewall, 1987.
   Second, determine the sexual
   development and fecundity of each mysid in each replicate. The effluent should be poured off in the same
   manner as during renewals. For each replicate remove the mysids and place each one in a separate well
   of a multi-well slide. Any excess water transferred with the mysid can be removed from the well to make
   viewing under a microscope easier.

   Using a stereomicroscope at 240X, determine the sexual development of each mysid and record it on
   the test data sheet (see Figure 4). This must be conducted while the mysids are alive because they turn
   opaque upon dying. Figures 6 through 9 illustrate the sexual characteristics used to determine the matu-
   rity and fecundity of the mysids.

   Figure 6 is a mature female with eggs in the oviducts. This is most easily determined when viewed from
   above and is determined by large, dark, oval-shaped bodies in the mid-section of the thorax.

   Figure 7 shows a mature female with eggs in the brood pouch, and is characterized by the presence of
   dark pigmented spots on the lateral sides of the body. These can be seen both from above and from
   the side. Females that have no eggs or embryos have an empty brood pouch and empty oviducts. These
   females can be identified by a single dark spot on each half of the brood pouch. These spots can be seen
   from both above and from the side, although from the top is easiest. The video provides examples of
   females with, and without, eggs and embryos.

   Figure 8 presents a mature male mysid. Males are determined by the presence of testes that appear either as
   clear circles, when viewing them from above, or as appendages at the junction  of the thorax and abdomen when
   viewing them from the side.

   Figure 9 presents a diagram of an immature mysid. Immature mysids are those that do not have character-
   istics that determine their classification as either mature males or females. Care must be taken, however,
   not to mistake a barren female for an immature rrtysid. As the sex of each mysid is determined it should be
   recorded on the survival and fecundity data sheet (see Figure 4).

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                               Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                      Supplement to Training Video

       After the sex, maturity, and
       fecundity of each mysid from
       one replicate is determined,
       all of the mysids from that
       replicate should be placed
       on a Nitex screen that rests antennuie
       on top of a beaker. Rinse the
       mysids with deionized water
       to remove any salts that may
       interfere with the dry weights.
       After the animals are rinsed
       they are placed on the desig-
       nated pre-tared piece of alu-
       minum foil for that replicate.
       Note that all of the mysids
       from one replicate are placed
       on the same piece of foil.
       Once this process has been
       repeated for all of the repli-
       cates the mysids are dried in
       an oven at 60C for 24 hours
       or 105C for at least six
       hours. The mysids must be
       completely dried before they
       are weighed but they should
       not be overdried.
       Figure 7. Mature Female A. bahia with Eggs in Oviducts and
       Developing Embryos in Brood Sac. Lateral view (top) Dorsal view
       (bottom)
                       eyestalk
                                 carapace
                                                               itatocyst
                                                                   telson
                                        developing brood sac
                                        oviducts with developing ova
                                                                      .telson
                                                                        uropod
       The mysids should be
       transported and stored in a
       desiccator when weighing
       them. This prevents mois-
       ture from reabsorbing into
       the mysids. The mysids are
       weighed, one replicate at a
       time, to the nearest milligram
       (0.001 g.). Because small dif-
       ferences in weight or appear-
       ance can easily change the
       test results, it is critical to
       record observations and
       measurements clearly and
       accurately. See Figure 5
       for a sample data sheet
       for recording weights. The
       minimum requirement for an
       acceptable test is an average
       weight of at least 0.20 mg/
       mysid in the controls.

       The analysis of this test com-
       pares the maturity, fecundity,
       growth, and survival of the
       Source: Lussier, Kuhn, and Sewall, 1987.

       Figure 8. Mature Male A. bahia. Lateral view (top) Dorsal View
       (bottom)
                      eyestalk
                                   carapace
antennuie
                                                               statocyst

                                                                    telson
                                                                      telson
                                                                        uropod
       Source: Lussier, Kuhn, and Sewall, 1987.
10

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                          Mysid (Americamysis bah/a) Survival, Growth, and Fecundity Toxicity Tests
                                                                                Supplement to Training Video
   exposed mysids to the con-
   trol mysids. The Saltwater
   Chronic Methods Manual
   (EPA, 2002a) provides
   instructions for statistical  antennule
   analysis of the survival,
   growth, and fecundity data.

   TEST ACCEPTABILITY
   AND DATA REVIEW
   Test data are reviewed to
   verify that EPA's WET test
   methods' acceptability
   criteria (TAG) requirements
   for a valid test have been
   met. For instance, the TAG
   requires 80% or greater
   survival in controls with an
   average weight of at least
   0.20  mg/mysid and 50%
   or more of the females in
   the controls must have
   eggs.
Figure 9. Immature A. bahla. Lateral view (top) Dorsal view
(bottom)
               eyestalk
                          carapace
                                                        statocyst
                                                            telson
                                                                telson
                                                                 uropod
Source: Lussier, Kuhn, and Sewall, 1987)
   The concentration-response relationship generated for each multi-concentration test must be reviewed to
   ensure that calculated test results are interpreted appropriately. In conjunction with this requirement, EPA
   has provided recommended guidance for concentration-response relationship review (EPA, 2000a).

   EPA's promulgated toxicity testing method manuals (EPA, 2002a, b) recommend the use of point estima-
   tion technique approaches for calculating endpoints for effluent toxicity tests under the NPDES program.
   The promulgated methods also require a data review of toxicity data and concentration-response data, and
   require calculating the percent minimum significant difference (PMSD) when point estimation (e.g., LC50,
   IC25) analyses are not used. EPA specifies the PMSD must be calculated when NPDES permits require sub-
   lethal hypothesis testing. EPA also requires that variability criteria  be applied as a test review step when
   NPDES permits require sub-lethal hypothesis testing endpoints (i.e., no observed  effect concentration
   [NOEC] or lowest observed effect concentration [LOEC]) and the effluent has been determined to have no
   toxicity at the permitted receiving water concentration (EPA, 2002b). This reduces the within-test variabil-
   ity and increases statistical sensitivity when test endpoints are expressed using hypothesis testing rather
   than the preferred point estimation techniques.
                                                                                                  II

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                 Supplement to Training Video
Citations and Recommended  References

       American Society for Testing and Materials. 1987. Practice Guide for Conducting Life Cycle Toxicity Tests
               with Saltwater Mysids. ASTM E-1191-87.  ASTM, Philadelphia, PA.

       American Public Health Association. 1985. Standard Methods for the Examination of Water and
               Wastewater. 16th Ed. APHA, Washington, D.C.

       Bahner, L.H., C.D. Craft, and D.R. Nimmo. 1975. A saltwater flow-through bioassay method with controlled
               temperature and salinity. Progr. Fish-Cult. 37:126-129.

       Borthwick, P.W.  1978. Methods for acute static toxicity tests with mysid shrimp (Mysidopsis bahia). In:
               Bioassay Procedures for the Ocean Disposal Permit Program. U.S. Environmental Protection
               Agency, Environmental Research Laboratory, Gulf Breeze, Florida.

       Breteler, R.J., J.W. Williams, and R.L. Buhl. 1982. Measurement of chronic toxicity using the opossum
               shrimp Mysidopsis bahia. Hydrobiol. 93:189-194.

       Buikema, A.L., B.R. Neiderlehner, and J. Cairns. 1982. Biological monitoring. Part IV. Toxicity Testing.
               Water Res. 16:239-262.

       EPA. 1991. Technical Support Document for Water Quality-based Toxics Control. U.S. EPA Office of Water
               Enforcement and Permits, Washington, D.C. EPA-505-2-90-001.

       EPA. 2000a. Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications
               Under the National Pollutant Discharge Elimination System Program. Office of Wastewater
               Management, Washington, D.C. EPA 833-R-00-003.

       EPA. 2000b. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR
               Part 136). Office of Water, Washington, D.C. EPA 821-B-00-004.

       EPA. 2002a. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
               Marine and Estuarine Organisms, Third Edition. (Saltwater Chronic Methods Manual). Office of
               Water, Cincinnati, OH. EPA-821-R-02-014.

       EPA. 2002b. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
               and Marine Organisms, Fifth Edition. (Acute Methods Manual). Office of Water, Cincinnati, OH.
               EPA-821-R-02-012.

       EPA. 2002c. Final Rule. 40 CFR Part 136. Guidelines Establishing Test Procedures for the Analysis of
               Pollutants; Whole Effluent Toxicity Test Methods. 67 FR 69952-69972, November 19, 2002.

       EPA. 2009a. Mysid (/Americamys/s bahia) Survival, Growth, and Fecundity Toxicity Tests. Supplement to
               Training Video. Whole Effluent Toxicity Training Video Series, Saltwater Series.  March 2009. EPA
               833-C-09-001.

       EPA. 2009b. Culturing Americamysis bahia. Supplement to Training Video. Whole Effluent Toxicity Training
               Video Series, Saltwater Series. March 2009. EPA 833-C-09-001.

       Heard, R.W. and W.W. Price. 2006. A Taxonomic Guide to the Mysids of the South Atlantic Bight. U.S.
               Department of Commerce, National Oceanic and Atmospheric Administration.

       Lussier, S.M., A. Kuhn and R. Comeleo. 1999. An evaluation of the 7-day toxicity test with Americamysis
               bahia (formerly Mysidopsis bahia). Environ. Toxicol. and Chem. 18:2888-2893. [Errata: in the sec-
               tion on Experimental Design, the test chamber should read "200-ml plastic cup" not "30-ml."]
12

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U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                               Supplement to Training Video
   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.

   Lussier, S.M., A. Kuhn, and J. Sewall. 1987. Guidance manual for conducting 7-day mysid survival/
           growth/reproduction study using the estuarine mysid, Mysidopsis bahia. Contribution No. X106.
           In: Schimmel, S.C., ed. Users guide to the conduct and interpretation of complex effluent toxic-
           ity tests at estuarine/marihe sites. Environmental Research Laboratory, U.S. Environmental
           Protection Agency, Narragansett, Rhode Island. Contribution No. 796,  265 pp.

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

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

   Nimmo, D.R., T.L. Hamaker, and C.A. Sommers. 1978. Entire life cycle toxicity test using mysids
           (Mysidopsis bahia) in flowing water. 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. 64-68.

   Personne, G., E. Jaspers, and C. Glaus, eds. 1980. Ecotoxicological testing for the marine environment.
           Vol.  1 State University of Ghent and Institute for Marine Scientific Research, Bredene, Belgium.

   Price, W.W., R.W. Heard and L. Stuck. 1994. Observations on the genus Mysidopsis sars. 1864 with the
           designation of a new genus, Americamys/s, and the descriptions of Americamysis alleni and A.
           stucki (Peracarida: Mysidacea: Mysidae), from the Gulf of Mexico. Proc. Biol. Soc. Wash. 107:680-
           698.

   Schimmel, S.C., ed. 1987. Users guide to the conduct and interpretation of complex effluent toxicity tests
           at estuarine/marine sites. Environmental Research  laboratory, U.S. Environmental Protection
           Agency, Narragansett, Rhode Island. Contribution No.  796, 265 pp.

   Stuck, K.C., H.M. Perry and R.W. Heard 1979. An annotated key to the Mysidacea of the North Central Gulf
           of Mexico. Gulf Res. Rept. 6(3):255 - 238.

   Walters, D.B. and C.W. Jameson. 1984. Health and safety for toxicity testing. Butterworth Publ., Woburn,
           Massachusetts.

   EPA references are available online at www.epa.gov/npdes.

   If you need additional copies of this document,  you can download it at:
           www.epa.gov/npdes/wqbasedpermitting.
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                                                                                                  Supplement to Training Video
                                                 Intentionally Left Blank
14

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                    Supplement to Training Video
Glossary
       Acute toxicity. An adverse effect measured on a group of test organisms during a short-term exposure in
               a short period of time (96 hours or less in toxicity tests). The effect can be measured in lethality
               or any variety of effects.

       Artem/a. The marine invertebrate (referred to as brine shrimp) used as the recommended food source for
               mysid cultures and test organisms; Brazilian or Colombian strains are preferred because the sup-
               plies are found to have low concentrations of chemical residues and nauplii are of suitably small
               size.

       Chronic toxicity. An adverse effect that occurs over a long exposure period. The effect can be lethality,
               impaired growth, reduced reproduction, etc.

       Cyst. The life stage of unhatched Artemia.

       Diluent water. Dilution water used to prepare the effluent concentrations.

       Effluent concentrations. Concentrations or dilutions of an effluent sample to which test organisms are
               exposed to determine the biological effects of the sample on the test organism.

       Effluent sample. A representative collection of the discharge that is to be tested.

       Fecundity. Productivity or fertility as measured in this test as the percentage of females.with eggs in the
               oviduct and/or brood pouch.

       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.

       Hypothesis testing.  Technique (e.g., Dunnett's test) that determines what concentration is statistically
               different from the control. Endpoints determined from hypothesis testing are NOEC and LOEC.

       IC25 (Inhibition Concentration, 25%). The point estimate of the toxicant concentration that would cause a
               25% reduction in a non-quantal biological measurement (e.g., reproduction or growth) calculated
               from a continuous model.

       LC50 (Lethal Concentration, 50%). The concentration of toxicant or effluent that would cause death to
               50% of the test organisms at a specific time of observations (e.g., 96-hour LC50).

       Lowest Observed Effect Concentration (LOEC). The LOEC is the lowest concentration of toxicant to
               which organisms are exposed in a test, which causes statistically significant adverse effects on
               the test organisms (i.e., where the values for the observed endpoints are statistically significantly
               different from the control). The definitions of NOEC and LOEC assume a strict dose-response
               relationship between toxicant concentration and organism response.

       Minimum Significant Difference (MSD). The MSD is the magnitude of difference from the control where
               the null  hypothesis is rejected in a statistical test comparing a treatment with a control. MSD
               is based on the number of replicates, control performance and power of the test. MSD is often
               measured as a percent and referred to as PMSD.

       Mysid (Amer/camys/s bahia). An estuarine crustacean, formerly known as Mysidopsis bahia, ranging 4.4
               mm to 9.4 mm in length found from the Gulf of Mexico and along the Atlantic coast as far north
               as Rhode Island, used in test procedures as an indicator species for marine or estuarine aquatic
               toxicity.

                                                                                             Glossary-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY         Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                     Supplement to Training Video
       Nauplii. Free-swimming microscopic larvae stage characteristic of copepods, ostracods, barnacles, etc.
               typically only with three pairs of appendages.

       No Observed Effect Concentration (NOEC).  The NOEC is the highest tested concentration of toxicant to
               which organisms are exposed in a full life-cycle or partial life-cycle (short-term) test, that causes
               no observable adverse effect on the test organism (i.e., the highest concentration of toxicant
               at which the values for the observed responses are not statistically significantly different from
               the controls). NOECs calculated by hypothesis testing are dependent upon the concentrations
               selected.

       NPDES (National Pollutant Discharge Elimination System) Program. The national program for issuing,
               modifying, revoking and reissuing, terminating, monitoring and enforcing permits, and imposing
               and enforcing pretreatment requirements, under Sections 307, 318, 402, and 405 of the Clean
               Water Act.

       Point Estimation Techniques. This technique is used to  determine the effluent concentration at which
               adverse effects (e.g., fertilization, growth or survival) occurred, such as Probit, Interpolation
               Method, Spearman-Karber. For example, a concentration at which a 25% reduction in
               reproduction and survival occurred.

       Receiving Water Concentration (RWC). The RWC is the  concentration of a toxicant or the parameter
               toxicity in the receiving water (i.e., riverine, lake, reservoir, estuary or ocean) after mixing.

       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 renewal. The exposure medium is replaced each day by moving the test animal to a new test cup
               prepared with the proper effluent concentration.

       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.

       Toxicity test. A test to measure the toxicity of a chemical or effluent using living organisms. The test
               measures the degree of response of an exposed  organism to a specific chemical or effluent.

       WET (Whole effluent toxicity). The total toxic effect of an effluent measured directly with a toxicity test.
Glossary-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                         Supplement to Training Video
Appendix A
Summary  of Test Conditions and Test Acceptability
Criteria
   Table A-l. Summary of Test Conditions and Test Acceptability Criteria for Americamys/s bahia 7-day
   Survival, Growth, and Fecundity Toxicity Test
Test type
Salinity
Temperature (C)
Photoperiod
Light intensity (quality)
Test chamber size
Test solution volume
Renewal of test solutions
Age of test organisms
Number of concentrations per study
Number of organisms per test chamber
Number of replicate chambers per concen-
tration
Source of food
Feeding regime
Aeration
Dilution water
Effects measured
Cleaning
Sample volume needed
Test concentrations
Dilution factor
Test duration
Endpoints
Test acceptability criteria
Sampling requirements
Static renewal (required)
20%o - 30%o  2%0 (recommended)
26  1 C (recommended) '
16 hours light; 8 hours dark, with phase on/off period (recommended)
10-20 uE/mVs (50 - 100 ft-c) (ambient lab levels) (recommended)
8 oz plastic disposable cups, or 400 mL glass beakers (recommended)
ISO mL per replicate cup (recommended minimum)
Daily (required)
7 days at start of test (required)
Minimum of 5 concentrations and a control (required minimum)
5 (40 per concentration) (required minimum)
8 (required minimum)
Newly hatched Anemia nauplii (<24-hr old; required)
Feed ISO 24-hr old nauplii per mysid daily, half after test solution
renewal and half after 8 - 12 hr (recommended)
None unless DO falls below 4.0 mg/L, then gently aerate all cups
(recommended)
Natural seawater, or hypersaline brine diluted with deionized water,
or artificial seasalts (available options)
Survival and growth (required); egg development (recommended)
Pipet excess food from cups daily immediately before test solution
renewal and feeding (recommended)
3 L per day (recommended)
Effluents: 5 and a control (required)
Receiving waters: 100% receiving water (or minimum of 5) and a con-
trol (recommended)
Effluents: S 0.5 series (required)
Receiving waters: None, or ^ 0.5 (recommended)
7 days (required)
Survival and growth (required); and egg development (recommended)
80% or greater survival, average dry weight 0.20 mg or greater in
controls (required); fecundity may be used if 50% or more of females in
controls produce eggs (required if fecundity endpoint used)
For on-site tests, samples collected daily and used within 24 hr of the
time they are removed from the sampling device. For off-site tests, a
minimum of three samples (e.g., collected on days one, three, and five)
with a maximum holding time of 36 hr before first use (see Saltwater
Chronic Methods Manual, Section 8, Effluent and Receiving Water
Sampling, Sample Handling and Sample Preparation for Toxicity Test,
Subsection 8.5.4) (required)
   Source: Adapted from EPA, 2002a.

'Lussier at al, 1999 found that test conducted at 26C - 27C exhibited higher probability of meeting test acceptability criteria for fecundity
than tests conducted at 26 \C.

                                                                                       A-l

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     U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                         Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                                       Supplement to Training Video
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A-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                           '                      Supplement to Training Video
Appendix  B
Apparatus and  Equipment  List
       Air line, and air stones. For aerating cultures, brood chambers, and holding tanks, and supplying air to
               test solutions with low DO.

       Air pump. For oil-free air supply.

       Balance. Analytical, capable of accurately weighing to 0.00001 g.

       Beakers or flasks. Six, borosilicate glass or non-toxic plasticware, 2 - 3 L for making test solutions.

       Brine shrimp (Artem/a) culture unit. See section on "Maintaining and Feeding Cultures."

       Depression glass slides or depression spot plates. Two for observing organisms.

       Desiccator. For holding dried organisms.

       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.

       Droppers, and glass tubing with fire polished edges. 4 mm inner diameter for transferring organisms.

       Drying oven. 50 - 105C, for drying organisms.

       Environmental chamber or equivalent facility with temperature control (26  1C).

       Facilities for holding and acclimating test organisms.

       Forceps (fine tips such as jewelers forceps). For transferring organisms to weighing boats.

       Light box. For illuminating organisms during examination.

       Meters: pH and DO, and specific conductivity. For routine physical and chemical measurements.

       Mysid (Americamysis bahia) culture unit. See section on "Maintaining and Feeding Cultures". The test
               requires a minimum  of 240 7-day old (juvenile) mysids.

       NITEX or stainless steel mesh sieves.  150 urn and 100 urn for concentrating organisms; 1 mm mesh
               and 300 urn mesh for collection of juveniles.

       Pipet bulbs and fillers. Propipet, or equivalent.

       Reference weights, Class S. For checking performance of balance.

       Refractometer or other method. For determining salinity.

       Samplers. Automatic sampler, preferably with sample cooling capability, that can collect a 24-hour
               composite sample of 5 L.

       Separatory funnels, 2-liters. Two to four funnels for culturing Artem/a.

       Standard or micro-Wlnkler apparatus. For determining DO and checking DO meters.
                                                                                                B-l

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    U S ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                    Supplement to Training Video
       Test vessels. 200 ml borosilicate glass beakers or 8 oz disposable plastic cups 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). To avoid potential
               contamination from the air and excessive evaporation of test solutions during the test, the
               chambers should be covered with safety glass plates or sheet plastic (6 mm thick).

       Thermometers, bulb-thermograph or electronic-chart type. For continuously recording temperature.

       Thermometers, glass or electronic, laboratory grade. For measuring water temperatures.

       Thermometers. National Bureau of Standards Certified (see EPA 2002a). Used to calibrate laboratory
               thermometers.

       Trays. For test vessels: one large enough to transport eight vessels at one time; one to hold 56 test
               vessels (approximately 90 x 48 cm).

       Volumetric flasks and graduate cylinders. Class A. Borosilicate glass or non-toxic plastic labware,
               50 - 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.

       Water purification system. Millipore Milli-Q deionized water or equivalent.
B-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americamysis bahio) Survival, Growth, and Fecundity Toxicity Tests
                                                                                 Supplement to Training Video
Appendix  C:   Reagents and  Consumable Materials

       Data sheets. One set per test for recording data

       Effluent, receiving water, and dilution water.  Dilution water containing organisms that might prey upon
               or otherwise interfere with the test organisms should be filtered through a fine mesh (with 150 pm
               or smaller openings).

               Saline test and dilution water. The salinity of the test water must be in the range of 20%o - 30%o.
               The salinity should vary by no more than 2%0 among the chambers on a given day. If effluent and
               receiving water tests are conducted concurrently, the salinities of these tests should be similar.

               It is important to maintain a constant salinity across all treatments during a test. It is desirable
               to match the test salinity with that of the receiving water. Two methods are available to adjust
               salinities - a hypersaline brine (HSB) derived from natural seawater or artificial sea salts. Both are
               described in EPA, 2002a.

       Food source. Feed the mysids Artemia  nauplii that are less than 24-hour-old.

       Laboratory quality assurance samples and standards

       Markers, waterproof. For marking containers, etc.

       Membranes and filling solutions for DO probe. Or reagents, for modified Winkler analysis
               (See EPA, 2002a).

       pH buffers 4, 7, and 10 -  (Or as  per instructions of instrument manufacturer) for standards and
               calibration check (see EPA 2002a).

       Reagent water Distilled or deionized water that does not contain substances which are toxic to the test
               organisms.

       Reference toxicant solutions. Reference toxicants such as sodium chloride (NaCI), potassium chloride
               (KCI), cadmium chloride (CdCI2), copper sulfate (CuS04), sodium dodecyl sulfate (SDS), and
               potassium dichromate (K2Cr207), are suitable for use in the NPDES Program and other Agency
               programs requiring aquatic toxicity tests.

       Sample containers. For sample shipment and storage.

       Tape, colored. For labeling test containers.

       Test organisms. The test is begun with  7-day-old juvenile Amer/camys/s bahia (mysids).

       Weighing pans, aluminum. To determine the dry weight of the organisms
                                                                                                C-l

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     U S ENVIRONMENTAL PROTECTION AGENCY          Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                                  Supplement to Training Video
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    U.S. ENVIRONMENTAL PROTECTION AGENCY        Mysid (Americomysis bohia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                   Supplement to Training Video
Appendix D:
Preparing  Hypersaline Brine  (HSB)
       Salinity adjustments are a vital part of using marine and estuarine species for toxicity testing. Because the
       majority of industrial and sewage treatment effluents entering marine and estuarine waters contain little or
       no measurable salts, the salinity of these effluents must be adjusted before exposing estuarine or marine
       plants and animals to the test solutions. It also is important to maintain constant salinity across all treat-
       ments throughout the test for quality control. Finally, matching the test solution's salinity to the expected
       receiving water's salinity may require salinity adjustments. NHEERL-AED uses HSB, prepared from filtered
       natural seawater, to adjust exposure solution salinities.

       , HSB has several advantages over artificial sea salts that make it more suitable for use in toxicity testing.
       Concentrated brine derived from natural seawater contains the necessary trace metals, biogenic colloids,
       and some of the microbial components necessary for adequate growth, survival, and/or reproduction of
       test organisms. HSB can  be held for prolonged periods without any apparent degradation, added directly to
       the effluent to increase the salinity, or used as control water by diluting to the desired salinity with deion-
       ized water. The brine can be made from any high-quality, filtered seawater supply through simple heating
       and aerating.

       GENERATING THE BRINE
       The ideal container for making brine from natural seawater has a high surface-to-volume ratio, is made of a
       non-corrosive material, and is easily cleaned. Shallow fiberglass tanks are ideal.

       Thoroughly clean the tank, aeration supply tube, heater, and any other materials that will be in direct
       contact with the brine before adding seawater to the tank. Use a good quality biodegradable detergent, fol-
       lowed by several thorough deionized-water rinses.

       Collect high-quality (and preferably high-salinity) seawater on an incoming tide to minimize the possibility of
       contamination. Special care should be used to prevent any toxic materials from coming in contact with the
       seawater. The water should be filtered to at least 10 urn before placing into the brine tank. Fill the tank with
       seawater, and slowly increase the temperature to 40C. If a heater is immersed directly into the seawater, make
       sure that the heater components will not corrode or leach any substances that could contaminate the brine. A
       thermostatically controlled heat exchanger made from fiberglass is suggested.

       Aeration prevents temperature stratification and increases the rate of evaporation. Use an oil-free air
       compressor to prevent contamination. Evaporate the water for several days, checking daily (or more or
       less often, depending on  the volume being generated) to ensure that the salinity does not exceed  100%o
       and the temperature does not exceed 40C. If these changes are exceeded, irreversible changes in the
       brine's properties may occur. One such change noted in original studies at NHEERL-AED was a reduction
       in the alkalinity of seawater made from brine with salinity greater than 100%0, and a resulting reduction in
       the animals' general  health. Additional seawater may be added to the brine to produce the volume of brine
       desired.

       When the desired volume and salinity of brine is prepared, filter the brine through a 1-mm filter and pump
       or pour it directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are most
       suitable). Cap the containers, and record the measured salinity and the date generated. Store the brine in
       the dark at room temperature.
                                                                                                   D-l

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     .S. ENVIRONMENTAL PROTECTION AGENCY
                                             Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests
                                                                                  Supplement to Training Video
      SALINITY ADJUSTMENTS USING HYPERSALINE BRINE
      To calculate the volume of brine (Vb) to add to a 0%o sample to produce a solution at a desired salinity (Sf),
      use this equation:
                                             vb * sb = s, * vf
      Where:
v,=
                    v,=
volume of brine, ml

salinity of brine, %o

final salinity, %0

final volume needed, ml
      Table D-l gives volumes needed to make 20%o test solutions from effluent (0%o), deionized water, and
      100%o MSB. The highest effluent exposure concentrations achievable are 80% effluent at 20%o salinity
      and 70% effluent at 30%o salinity. Test solutions presented in Table D-l are not meant as recommenda-
      tions, rather as examples.
   Table D-l. Preparation of Test Solutions at a Salinity of 20%o Using HSB for a Final Test Concentration
   Volume of 2000 ml.
Exposure Concentration
(% effluent)
80
40
20
10
5
Control
Effluent
(assumes 0%o salinity)
(mL)
1,600
800
400
200
100

De/onized
Water (mL)
0
800
1,200
1,400
1,500
2,000
HSB
(f 00%. salinity) (mL)
400
400
400
400
400
400
D-2

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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WHOLE EFFLUENT ToxiCITY  TRAINING VIDEO SERIES  saltwater series
          Sperm Cell Toxicity
          Tests Using the Sea Urchin
          (Arbacia punctulata)
          Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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                            NOTICE

    The revision of this guide has been funded wholly or in part by the
Environmental Protection Agency under Contract EP-C-05-063. Mention of
trade names or commercial products does not constitute endorsement or
                     recommendation for use.

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^,| U.S. ENVIRONMENTAL PROTECTION AGENCY            Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                   Supplement to Training Video
Foreword
       This guide serves as a supplement to the video "Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia
       punctulata" (EPA, 2009). The methods illustrated in the video and described in this supplemental guide
       support the methods published in the U.S. Environmental Protection Agency's (EPA's) Short-term Methods
       for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms,
       Third Edition (EPA, 2002a), referred to as the Saltwater Chronic Methods Manual. The video and this guide
       provide details on preparing for and conducting the test based on the expertise of personnel at the follow-
       ing EPA Office of Research and Development (ORD) laboratories:

           National Health and Environmental Effects Research Laboratory (NHEERL) - Atlantic Ecology Division
           in Narragansett, Rhode Island

           NHEERL - Gulf Ecology Division in Gulf Breeze, Florida

           National Exposure Research Lab (NERL) - Ecological Exposure Research Division (EERD) in
           Cincinnati, Ohio

       This guide and its accompanying video are part of a series of training videos produced by EPA's Office of
       Wastewater Management. This Saltwater Series includes the following videos and guides:

           "Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests"

           "Culturing Americamysis bahia"

           "Sperm Cell  Toxicity Tests Using the Sea Urchin, Arbacia punctulata"

           "Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests"

           "Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
               and Growth Toxicity Tests"

       The Freshwater Series, released in 2006, includes the following videos and guides:

           "Ceriodaphnia Survival and Reproduction Toxicity Tests"

           "Culturing of Fathead Minnows (Pimephales promelas)"

           "Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests"

       All of these videos are available through the National Service Center for Environmental Publications
       (NSCEP)  at 800 490-9198 or nscep@bps-lmit.com.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                       Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulota)
                                                                                             Supplement to Training Video
                                             Intentionally Left Blank

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    U.S. ENVIRONMENTAL PROTECTION AGENCY            Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                            Supplement to Training Video
Contents
      Foreword	i
      Introduction	1
      Background	:	1
      Water and Light	1
      Obtaining and Maintaining Sea Urchins	1
      Culture Water	1
      Photoperiod	1
      Culture Vessels	2
      Water Delivery Systems	2
      Food Preparation	2
      Test Method	2
      Obtaining Gametes	2
      Making Stock Solutions of Sperm and Eggs	3
      Effluent Preparation	5
      Beginning the Test	5
      Routine Chemistries	6
      Terminating the Test	6
      Test Acceptability and Data Review	6
      Citations and Recommended References	7
      Glossary	Glossary-1
      Appendix A: Preparing Hypersaline Brine (MSB)	A-l
      Appendix B: Apparatus and Equipment	B-l
      Appendix C: Reagents and Consumable Materials	C-l
      Appendix D: Summary of Test Conditions and Test Acceptability Criteria	D-l
      Appendix E: Data Sheets	E-l

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U.S. ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)'
                                                                            Supplement to Training Video
   FIGURES
   Figure 1. Schematic of the aboral surface of Arbacia punctulata, with spines partly removed to
   show structure, especially the genital pores	2
   Figure E-l. Sperm Cell Toxicity Test, Sample Data Sheet #1	E-l
   Figure E-2. Sperm Cell Toxicity Test, Sample Data Sheet #2 - Raw Data	E-2
   TABLES
   Table 1. Fifty Percent Serial Dilution Method for Counting Sperm Cell Density	3
   Table A-l. Preparation of Test Solutions at a Salinity of 30%o Using MSB for a Final Test
   Concentration Volume of  1000 ml	A-2
   Table A-2. Preparation of Test Solutions at a Salinity of 30%o Using Natural Seawater, Hypersaline
   Brine, or Artificial Sea Salts	A-2

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    U S ENVIRONMENTAL PROTECTION AGENCY            Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                  Supplement to Training Video
Introduction

       This supplemental guide accompanies the Environmental Protection Agency's (EPA's) video training for
       conducting sea urchin (Arbacia punctulata) fertilization toxicity tests (EPA,. 2009). The test method is found
       in Section  15 of EPA's Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
       Waters to Marine and Estuarine Organisms, Third Edition (EPA, 2002a). The test was developed at EPA's
       Office of Research and Development's (ORD's) National Health and Environmental Effects Research
       Laboratory-Atlantic Ecology Division (NHEERL-AED) in Narragansett, Rhode Island, and is based on the
       freshwater tests developed at the EPA Mid-Continent Ecology Division (MED) in Duluth, Minnesota. The
       material presented in both the video and this guide summarizes the methods but does not replace a thor-
       ough review and understanding of the methods by laboratory personnel before conducting the test.
Background
       Under the National Pollutant Discharge Elimination System (NPDES) program (Section 402 of the Clean
       Water Act), EPA uses toxicity tests to monitor and evaluate effluents for their toxicity to biota and their
       impact on receiving waters. By determining acceptable or safe concentrations for toxicants discharged
       into receiving waters, EPA can establish NPDES permit limitations for toxicity. These WET (Whole effluent
       toxicity) permit limitations regulate pollutant discharges on a whole effluent effect basis rather than by a
       chemical-specific approach only.

       Whole effluent toxicity methods measure the synergistic, antagonistic, and additive effects of all the chemi-
       cal, physical, and additive components of an  effluent that adversely affect the physiological and biochemi-
       cal functions of the test organisms. Therefore, healthy organisms and correct laboratory procedures are
       essential for valid test results. Laboratory personnel should be very familiar with the test methods and with
       sea urchin handling techniques before conducting a test.

       This supplemental guide covers the procedures for conducting the test according to EPA's promulgated
       methods (40 CFR Part 136; EPA, 2002c) and also provides some helpful information that is not  presented
       in the Saltwater Chronic Methods Manual (EPA, 2002a).

       This test method examines the effect of effluent or receiving waters on the reproduction of sea urchin
       gametes after exposure in a static system for 1 hour and 20 minutes. Sperm cells are exposed to a series
       of effluent concentrations for 1 hour. The eggs are then introduced to the test chambers which contain
       the sperm cells. After 20 minutes, the test is ended and the effects on exposed gametes are compared to
       controls to determine if the effluent concentrations had any effect on fertilization.

       This guide and the accompanying video describe how the test is set up, initiated, terminated, and reviewed,
       including suggestions on maintaining healthy cultures of test animals.

Water and Light

       OBTAINING AND MAINTAINING SEA URCHINS
       Before conducting tests, healthy sea urchin cultures should be established. Adult sea urchins can be
       ordered from commercial biological supply houses, or collected along the Atlantic coast. Keep male and
       female animals in separate tanks. To determine the sex of each animal, briefly stimulate each with a
       12-volt transformer. This causes the immediate release of masses of gametes from genital pores on the
       top of the animal. The eggs are red and the sperm are white. Separate the animals into 20 L aerated fiber-
       glass tanks; each can hold about 20 adults.

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  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                 Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                 Supplement to Training Video
     CULTURE WATER
     The quality of water used for
     maintaining sea urchins is very
     important. Culture water and
     all water used for washing and
     dilution steps and for control
     water in the tests should be
     maintained at a salinity of
     30%o  2%o using natural
     seawater, hypersaline brine
     (MSB), or artificial sea salts.
     Instructions for making dilution
     water and MSB are provided in
     Appendix A of this document
     and Section 7 of the Saltwater
     Chronic Methods Manual (EPA,
     2002a).

     PHOTOPERIOD
                                Figure 1. Schematic of the aboral surface of Arbacia punctulata,
                                with spines partly removed to show structure, especially the genital
                                pores
                                                                           anus
                                                                                       suranal
                                                                                       plate
                                                                                         ocular
                                                                                         plate
                                                                                            genital
                                                                                            plate
                                                                                      genital
                                                                                      pore
                                       madreporite
                                                                   madreporic
                                                                   plate
The sea urchin conditions
should include a photoperiod
of 16 hours light and 8 hours
darkness. The light quality and
intensity should be at ambient laboratory levels, which is approximately 10 - 20 E/um2/s or 50 to 100 foot
candles (ft-c) (EPA, 2002a).

CULTURE VESSELS
Adult sea urchins are kept in natural or artificial seawater in a flow-through or recirculating aerated 40-L
glass aquarium.

Allow filtered seawater to flow into the tanks at a rate of 5 L per minute and maintain the temperature at
15C3C.

WATER DELIVERY SYSTEMS
Equip the adult sea urchin aquarium with an under-gravel or outside biological filter, or cartridge filter. A
stock of at least 12 males and 12 females are needed for routine testing. If the animals will be used  for an
on-site test, transport them separated by sex in separate or partitioned coolers packed with wet kelp and
paper towels. Once on site, the sea urchins should be transferred into separate 10-gallon aquarium tanks
with gravel-bed filtration. Even with filtration, the water should be changed periodically to maintain good
water quality.
Collect eggs first to avoid any
possible pre-fertilization.
                                   FOOD PREPARATION
                              Sea urchins are fed kelp of the species Laminaria obtained from uncori-
                              taminated coastal waters or ordered from commercial supply houses, or
                              romaine lettuce. Supply the urchins with ample food, renewing the kelp
each week and removing decaying kelp as necessary. Healthy sea urchins will attach to kelp or aquarium
walls within hours  any unhealthy animals should be removed and should not be used for testing. Every 1
to 2 weeks, empty and clean the tanks.

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                 Sperm Cell Toxicity Tests Using the Sea Urchin (Arfaocia punctulata)
                                                                                 Supplement to Training Video
    At AED, staff use the data
    sheet included in Appendix E for
    calculating and recording dilutions
Test Method

       OBTAINING GAMETES
       To prepare for the test, all vials, pipets, and pipet tips should be soaked in clean, 30%o seawater overnight.
       Collect eggs and sperm from healthy animals by transferring the animals into a shallow bowl filled with
       enough control seawater to just cover their shells. Eggs are obtained from female sea urchins using electri-
                                        cal stimulation by touching the shells close to the genital pores with
                                        electrodes from a 10 - 12-volt transformer for about 30 seconds.
                                        The red eggs pool on the sea urchin shell above the genital pores.
                                        These are collected from the shell using a 10 ml disposable syringe
                                        with an 18-gauge, blunt-tipped needle with the tip cut off so that it
                                        will rest on the shell without puncturing it. After collection, the needle
       is removed and the eggs emptied into conical centrifuge tubes. Pool the eggs and keep them at room tem-
       perature until  use, but not longer than a few hours. Four females should yield enough eggs to test five test
       dilutions plus one control, with four replicates.

       Obtain sperm from four male sea urchins. Again, place the animals in a shallow bowl with their shells barely
       covered with control seawater. Like the females, the males are induced to spawn by placing electrodes
       from a 10 - 12-volt transformer against their shells for 30 seconds. The sperm appear white. Collect the
       concentrated sperm that pools on top of the shell using a syringe fitted with an 18-gauge, blunt-tipped
       needle. Pool the sperm, keep the sample on ice, and record the collection time. The sperm must be used in
       a toxicity test within 1 hour of collection.

       MAKING STOCK SOLUTIONS OF SPERM AND EGGS
       To ensure reproducibility in the test results, the sperm and eggs must be concentrated to known dilutions
       using the 30%o seawater. During the exposure period, 2,500 sperm should be present for every one egg.
       Figure E-l, presented in Appendix E, provides a sample data sheet used to calculate the sperm and egg
       deliveries.

       After collection, the sperm should be in a volume of about 0.5 to 1 mL of control water in the collecting
       syringe. This is called the "sperm stock" solution. Perform a 50 percent serial dilution for counting the
       sperm cell density using the following dilution method (see Table 1).

       Add sperm from Vial E to both sides of a Neubauer hemacytometer. Let the sperm settle 15  minutes. Count
       the number of sperm in the central 400 squares on both sides of the hemacytometer under a compound
       microscope (100X).

       The average of the two sperm cell counts (sperm/mL or SPM) from Vial E = # x 104.

       Calculate the SPM in all the other suspensions based on this count:
                                           Vial A = 40 x SPM of Vial E
                                           Vial B = 20 x SPM of Vial E

                                           Vial D = 5xSPMofVial E
                                    SPM of original sample = 2000 x SPM of Vial E
The egg solution can be prepared
during the first hour of the test after
the sperm exposure has started.
     To prepare the sperm suspension for the test, select the vial containing an SPM greater than 5 x 107 SPM.
     To determine the dilution needed for the test:
                                  The calculated SPM
                                       (5 x 107)

                                     [(DF) x 10] -10
                                                           = Dilution Factor (DF)

                                                           = mL of seawater to add to selected vial

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                 Supplement to Training Video
         Table 1. Fifty Percent Serial Dilution Method for Counting Sperm Cell Density.
'
2.
3.
4.
5.
6.
7.
Add 400 uL of sperm stock to 20 mL of seawater to create Vial A.
using a 5-mL pipettor, or by inversion.
Add 10 ml from Vial A to 10 ml of seawater to create Vial B. Mix
5-mL pipettor, or by inversion.
Add 10 mL from Vial B to 10 mL of seawater to create Vial C. Mix
5-mL pipettor, or by inversion.
Add 10 mL from Vial C to 10 mL of seawater to create Vial D. Mix
a 5-mL pipettor, or by inversion.
Mix by gently pipetting
by gently pipetting using a
by gently pipetting using a
by gently pipetting using
Discard 10 mL from Vial D so that all vials now contain 10 mL.
Vial C is used to create a final dilution that is killed and counted. Add 10 mL 10% acetic acid
in seawater to Vial C; cap the vial and mix by inversion.
Add 1 mL of the killed sperm in Vial C to 4 mL of seawater in Vial
using a 4-mL pipettor.
E. Mix by gently pipetting
 1. 400 Ml
 sperm stock
                          2. 10ml
                                             3. 10 ml
                                                               4. 10 ml
                                                                                5. Discard 10 ml
                 Vial A
               20 mL stock
                seawater
  Vial B    7 1 m/    Vial C
10 ml stock     /    lOmLstock
 seawater    /       seawater
  VialD
10 mL stock
 seawater
            6.  10 ml 10% acetic
            acid in saltwater
                                                                   Mix well
                                                                   before each
                                                                   transfer.
                                    Vial C
     The sperm cell count in the test stock should be confirmed. Add 0.1 mL of test stock to 9.9 mL of 10
           percent acetic acid in seawater and count the sperm cells using a hemacytometer. This count
                        should average 50 5 cells. Only about 2.5 mL of sperm test stock solution is
                        needed for testing 5 test solutions and a control, with 4 or more replicates. Hold the
                        test stock on ice until the test begins, but no longer than 1 hour.

                        The eggs must be washed before preparing the standard egg dilution needed for
                        the test (2,000 eggs/mL). To wash the eggs, first remove the supernatant water
   from the settled eggs. Add seawater and mix carefully by inversion. Spin the  vial in a tabletop centrifuge at
   the lowest possible setting (e.g., 500xg) for 3 minutes to form a lightly packed  pellet. Wash and spin the
   eggs twice more. If at any time the wash water appears red the eggs are lysing (the membranes have been
   disturbed) and the eggs are unsuitable for testing; discard these eggs and start again.

   After washing, transfer the washed eggs to a beaker containing 200 mL of control seawater. This is called
   the "egg test stock." Mix the stock solution using gentle aeration until the egg solution is homogenous. The
   aeration device used in Narragansett  is a 3-pronged diffuser attached by flexible tubing to an air pump.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                               Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                               Supplement to Training Video
   Make a 1:10 dilution of the test stock for the purpose of counting the eggs. Cut the point from a wide-
   mouth pipet tip to make sure the eggs will not be damaged and transfer 1 ml of egg solution to a vial
   containing 9 ml of control water. Mix by inversion.

   Transfer 1 ml of the egg solution to a Sedgewick-Rafter counting chamber. Count the number of eggs
   under a dissecting microscope at 25X magnification. Ten times the number of eggs in that milliliter equals
   the number of eggs/mL  in the egg stock. The target concentration for test initiation is 2,000 eggs/ml.

   If the egg count is greater than or equal to 200 eggs, add the proper volume of water:

                      (# of eggs counted) - 200 = volume (ml) of control water to add
   If less than 200 eggs were
   counted, allow the eggs to
   settle in the beaker, remove
   the supernatant water to con-
   centrate the eggs to greater
   than 200, repeat the count,
   and dilute the egg test stock
   as described above.

   Verify the concentration by
   counting 1 ml of a 1:10
   dilution of the adjusted stock
   solution. The count for the
   final dilution should equal
   100  20 eggs/mL. The test
   requires 24 ml of egg test
   stock for a control and five
   exposure concentrations.

   EFFLUENT PREPARATION
Dilution Water
The type of dilution water used to make the test concentrations ,>s
dependent on the objectives of the test Any specific requirements
included in NPDES permits should be followed.  The Saltwater Chronic
Methods Manual (Section 7) provides the following guidelines:
*  If the test is conducted to estimate the absolute
of the effluent, synthetic dilution water should be used. If the1 cultures
were maintained in different water than used for dilution wafer, a
second set of control replicates should be conducted using the culture
water.
  If the test is conducted to estimate the chronic toxicity of the
effluent in uncontaminated receiving waters, the test con be
conducted using a grab sample of the receiving waters collected outside
the influence of the outfall, other uncontaminated waters, or standard
dilution water with the same salinity as the receiving waters, if the
cultures were maintained in different water than used for dilution water,
a second set of control replicates should be conducted using the culture
                                   If the test is conducted to estimate the additive or mitigating
                                 effects of the effluent on already contaminated receiving
                                 waters, the test must be conducted using receiving waters collected
                                 outside the influence of the outfall. Controls should be conducted us/'r
                                 both receiving water and culture water.
Effluent sampling should
be conducted according to
Section 8 of the Saltwater
Chronic Methods Manual
(EPA, 2002a) and any
specific requirements of a
NPDES permit. The effluent
or receiving waters should be
held at 0C - 6C until used
for testing. Under the NPDES
program, lapsed time from
sample collection to first use in the test must not exceed 36 hours. Under special conditions or variances,
samples may be held longer but should never be used for testing if held for more than 72 hours.

Maintain the salinity of the test samples to 30%o  2%o. To do this, effluent samples may need to be
adjusted using hypersaline brine (MSB). A recipe for MSB is provided in Appendix A of this manual.

Approximately 1 hour before the test is to begin, adjust approximately 1 L of effluent to the test tempera-
ture of 20C  1C and maintain that temperature while preparing the test concentrations. To test a series
of decreasing concentrations of effluent, use a dilution factor of > 0.5. When starting with effluent that
has 0%o salinity and using MSB, the maximum effluent concentration that can be prepared at 30%o is 70
percent effluent. Table A-l presents the volumes needed for the test concentrations using MSB.

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    U'S- ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                  Supplement to Training Video
       BEGINNING THE TEST
       In Narragansett, disposable glass vials are used as test chambers. They are labeled with concentration
       and replicate numbers and arranged in the partitioned cardboard box in which they are shipped. Prepare
       the effluent dilutions for four replicates of each concentration and the control solution to reduce variability
       among replicates. Each concentration should be prepared in one beaker and 5 ml distributed to each of the
       test chambers. Be sure the effluent temperature has been brought up to 20C before beginning the test.

       Within 1 hour of collecting and preparing the sperm test stock, add 100 pL of the well-mixed sperm test
       stock to each test and control vial. Cover the chambers, record the time, and maintain the chambers at
       20C + 1C for 1 hour.

       At the end of the hour, mix the egg test stock using gentle aeration and add 1 mL of the egg solution to
       each exposure vial using a wide-mouth pipet. When all of the vials contain eggs, lift the storage box and
       gently move it in circles to "swirl" the egg-sperm suspension. Cover the chambers, record the time, and
       incubate the eggs and sperm at 20C + 1C for 20 minutes.

       ROUTINE  CHEMISTRIES
       At the beginning of the exposure period, DO, pH, temperature and salinity are  measured in one chamber at
       each test concentration and the control.

       TERMINATING THE TEST
       After 20 minutes, end the test and preserve the samples by adding 2 mL of 1% formalin in seawater to
       each vial. .Cap the vials and record the time.  The test should be evaluated immediately but can be evalu-
       ated up to 48 hours later.
Test Acceptability and Data Review
       This test demonstrates the effluent or receiving water's effect on sea urchin fertilization. To evaluate this,
       exposed and control eggs are examined under a microscope and the number of unfertilized eggs in each
       test chamber is recorded.

       For each replicate, transfer about 80 - 120 uL of the preserved eggs to a multiple-chamber counting slide.
       If a Sedgewick-Rafter counting chamber is used, transfer about 1 mL. Using a compound microscope at
       100X magnification, observe 100 - 200 eggs per sample. This should be done with adequate ventilation,
       preferably under a hood, to reduce exposure to the formalin fumes.

       For each test chamber, record the total number of eggs counted, and the number that were not fertilized.
       Fertilized eggs are surrounded by a fertilization membrane, while unfertilized eggs lack this membrane.
       Abnormal eggs are not counted. Figure E-2  in Appendix E provides a  sample data collection sheet.

       Test data are reviewed to verify that test acceptability criteria (TAG) requirements for a valid test have been
       met. For the test to be acceptable, the control chambers are required to  have between 70% and 90% fer-
       tilization of the eggs. The concentration-response relationship generated for each multi-concentration test
       must be reviewed to ensure that calculated test results are interpreted appropriately. In conjunction with
       this requirement, EPA has provided recommended guidance for concentration-response relationship review
       (EPA, 2000b).

       EPA's promulgated toxicity testing method manuals (EPA, 2002a, b)  recommend the use of point estima-
       tion technique approaches for calculating endpoints for effluent toxicity tests under the NPDES program.
       The promulgated methods also require a data review of toxicity data and concentration-response data, and
       require calculating the percent minimum significant difference  (PMSD) when point estimation (e.g., LC50,
       IC25) analyses are not  used. EPA specifies the PMSD must be calculated when NPDES permits  require sub-
       lethal hypothesis testing. EPA also requires that variability criteria be applied as a test review step when

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    U.S. ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                  Supplement to Training Video
       NPDES permits require sub-lethal hypothesis testing endpoints (i.e., no observed effect concentration
       [NOEC] or lowest observed effect concentration [LOEC]) and the effluent has been determined to have no
       toxicity at the permitted receiving water concentration (EPA, 2002b). This reduces the within-test variabil-
       ity and increases statistical sensitivity when test endpoints are expressed using hypothesis testing rather
       than the preferred point estimation techniques.

       The sea urchin sperm cell test is currently used to assess the potential toxic effects of complex chemical
       mixtures on marine and estuarine organisms. Used in conjunction with chemical-specific methods, this test
       can provide a comprehensive and effective approach to assessing the impact of complex effluents dis-
       charged to the marine and estuarine environments.

Citations and  Recommended References

       EPA. 1991. Technical Support Document for Water Quality-based Toxics Control. U.S. EPA Office of Water
               Enforcement and Permits, Washington, D.C. EPA-505-2-90-001.

       EPA. 2000a. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR
               Part 136). Office of Water, Washington, D.C. EPA 821-B-00-004.

       EPA. 2000b. Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications
               Under the National Pollutant Discharge Elimination System Program. Office of Wastewater
               Management, Washington, D.C. EPA 833-R-00-003.

       EPA. 2002a. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
               Marine and Estuarine Organisms, Third Edition. (Saltwater Chronic Methods Manual). Office of
               Water, Cincinnati, OH. EPA-821-R-02-014.

       EPA. 2002b. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
               and Marine Organisms, Fifth Edition. (Acute Methods Manual). Office of Water, Cincinnati, OH.
               EPA-821-R-02-012.

       EPA. 2002c. Final Rule. 40 CFR  Part 136. Guidelines Establishing Test Procedures for the Analysis of
               Pollutants; Whole Effluent Toxicity Test Methods. 67 FR 69952-69972, November 19, 2002.

       EPA. 2009. Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata). Supplement to Training
               Video. Whole Effluent Toxicity Training Video Series, Saltwater Series. March 2009. EPA 833-C-
               09-001.

       EPA references are available online atwww.epa.gov/npdes.

       If you need additional copies of this document, you can download it at:
               www.epa.gov/npdes/wqbasedperrnitting.

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    U'S' ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
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Glossary
       Acute toxicity. An adverse effect measured on a group of test organisms during a short-term exposure in
               a short period of time (96 hours or less in toxicity tests). The effect can be measured in lethality
               or any variety of effects.

       Arbacia punctulata. A species of Arbacia genus of purple-spined sea urchins. Its natural habitat is in the
               Western Atlantic Ocean. Arbacia punctulata can be found in shallow water from Massachusetts
               to Cuba and the Yucatan Peninsula, from Texas to Florida in the Gulf of Mexico, the coast from
               Panama to French Guiana and in the Lesser Antilles, usually on rocky, sandy, or shelly bottoms.

       Chronic toxicity.  An adverse effect that occurs over a long exposure period. The effect can be lethality,
               impaired  growth, reduced reproduction, etc.

       Diluent water. Dilution water used to prepare the effluent concentrations.

       Effluent concentrations. Concentrations or dilutions of an effluent sample to which test organisms are
               exposed to determine the biological effects of the sample on the test organism.

       Effluent sample.  A representative collection of the discharge that is to be tested.

       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.

       Hypothesis testing. Technique (e.g., Dunnett's test) that determines what concentration is statistically
               different from the control. Endpoints determined from hypothesis testing are NOEC and LOEC.

       IC2s (Inhibition Concentration, 25%). The point estimate of the toxicant concentration that would cause a
               25% reduction in a  non-quantal biological measurement (e.g., reproduction or growth) calculated
               from a continuous model.

       Laminaria. The scientific name for a species of kelp given as food to laboratory sea urchins.

       LCSO (Lethal Concentration, 50%). The concentration  of toxicant or effluent that would cause death to
               50% of the test organisms at a specific time of observations (e.g., 96-hour LC50).

       Lowest Observed Effect Concentration (LOEC). The LOEC is the lowest concentration of toxicant to
               which organisms are exposed in a test, which causes statistically significant adverse effects on
               the test organisms  (i.e., where the values for the observed endpoints are statistically significantly
               different from the control). The definitions of NOEC and LOEC assume a strict dose-response
               relationship between toxicant concentration and organism response.

       Minimum Significant Difference (MSD). The MSD is the magnitude of difference from the control where
               the null hypothesis  is rejected in a statistical test comparing a treatment with a control. MSD
               is based on the number of replicates, control performance and power of the test. MSD is often
               measured as a percent and referred to as PMSD.

       No Observed Effect Concentration (NOEC).  The NOEC is the highest tested concentration of toxicant to
               which organisms are exposed in a full life-cycle or partial life-cycle (short-term) test, that causes
               no observable adverse effect on the test organism (i.e., the highest concentration of toxicant
               at which the values for the observed responses are not statistically significantly different from
               the controls). NOECs calculated by hypothesis testing are dependent upon the  concentrations
               selected.
                                                                                             Glossary-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
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       NPDES (National Pollutant Discharge Elimination System) Program. The national program for issuing,
               modifying, revoking, and reissuing, terminating, monitoring and enforcing permits, and imposing
               and enforcing pretreatment requirements, under Sections 307, 318, 402, and 405 of the Clean
               Water Act.

       Point Estimation Techniques. This technique is used to determine the effluent concentration at which
               adverse effects (e.g., fertilization, growth or survival) occurred, such as Probit, Interpolation
               Method, Spearman-Karber. For example, a concentration at which a 25% reduction in
               reproduction and survival occurred.

       Receiving Water Concentration (RWC). The RWC is the concentration of a toxicant or the parameter
               toxicity in the receiving water (i.e., riverine, lake, reservoir, estuary or ocean) after mixing.

       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.

       Toxicity test. A procedure to measure the toxicity of a chemical or effluent using living organisms. The
               test measures the degree of response of an exposed organism to a specific chemical or effluent.

       WET (Whole effluent toxicity). The total toxic effect of an effluent measured directly with a toxicity test.
Glossary-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                   Supplement to Training Video
Appendix A:
Preparing  Hypersaline  Brine (HSB)
       Salinity adjustments are a vital part of using marine and estuarine species for toxicity testing. Because the
       majority of industrial and sewage treatment effluents entering marine and estuarine waters contain little or
       no measurable salts, the salinity of these effluents must be adjusted before exposing estuarine or marine
       plants and animals to the test solutions. It also is important to maintain constant salinity across all treat-
       ments throughout the test for quality control. Finally, matching the test solution's salinity to the expected
       receiving water's salinity may require salinity adjustments. NHEERL-AED uses HSB, prepared from filtered
       natural seawater, to adjust exposure solution salinities.

       HSB has several advantages over artificial sea salts that make it more suitable for use in toxicity testing.
       Concentrated brine derived from natural seawater contains the necessary trace metals, biogenic colloids,
       and some of the microbial components necessary for adequate growth, survival, and/or reproduction of
       test organisms. HSB can  be held for prolonged periods without any apparent degradation, added directly to
       the effluent to increase the  salinity, or used as control water  by diluting to the desired salinity with deion-
       ized water. The brine can be made from any high quality, filtered seawater supply through simple heating
       and aerating.

       GENERATING THE BRINE
       The ideal container for making brine from natural seawater has a high surface-to-volume ratio, is made of a
       non-corrosive material, and is easily cleaned. Shallow fiberglass tanks are ideal.

       Thoroughly clean the tank, aeration supply tube, heater, and  any other materials that will be in direct
       contact with the brine before adding seawater to the tank. Use a good quality biodegradable detergent,  fol-
       lowed by several thorough deionized-water rinses.

       Collect high-quality (and preferably high-salinity) seawater on an incoming tide to minimize the possibility of
       contamination. Special care should be used to  prevent any toxic materials from coming in contact with the
       seawater. The water should  be filtered to at least 10 urn before placing into the brine tank. Fill the tank with
       seawater, and slowly increase the temperature to 40C. If a heater is immersed directly into the seawater, make
       sure that the heater components will not corrode or leach any substances that could contaminate the brine. A
       thermostatically controlled heat exchanger made from fiberglass is suggested.

       Aeration prevents temperature stratification and increases the rate of evaporation. Use an oil-free air
       compressor to prevent contamination. Evaporate the water for several days, checking daily (or more or
       less often, depending on  the volume being generated) to ensure that the salinity does not exceed 100%o
       and the temperature does not exceed 40C. If these changes are exceeded, irreversible changes in the
       brine's properties may occur. One such change noted in original  studies at NHEERL-AED was a reduction
       in the alkalinity of seawater made from brine with salinity greater than 100%o, and a resulting reduction in
       the animals' general health. Additional seawater may be added to the brine to produce the volume of brine
       desired.

       When the desired volume and salinity of brine is prepared, filter the brine through a 1-mm filter and pump
       or pour it directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are most
       suitable). Cap the containers, and record the measured salinity and the date generated. Store the brine  in
       the dark at room temperature.
                                                                                                   A-1

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      .S. ENVIRONMENTAL PROTECTION AGENCY
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                                                                                            Supplement to Training Video
        SALINITY ADJUSTMENTS USING HYPERSALINE BRINE
        To calculate the volume of brine (Vb) to add to a 0%o sample to produce a solution at a desired salinity (Sf),
        use this equation: .
        Where:
                      sf =
                      vf =
                 w  * c
                 vb    3t
volume of brine, ml

salinity of brine, %o

final salinity, %o

final volume needed, ml
        Table A-l presents volumes needed to make 30%o test solutions from effluent (0%o), deionized water, and
        100%o MSB. At 30%o salinity, the highest achievable concentration is 70% effluent.


    Table A-l. Preparation of Test Solutions at a Salinity of 30%o Using MSB for a Final Test Concentration
    Volume of 1000 ml.
Exposure
Concentration (%)
70
25
7
2.5
0.7
Control
Effluent
(0 %o)
(mL)
700
250
70
25
7

Deionized Water
(mL)

450
630
675
693
1,000
Hypersaline Brine
(I00%o)
(mL)
300
300
300
300
300

        Table A-2 gives examples of attainable exposure concentrations and dilution volumes needed when an
        effluent salinity is raised to 30%o using artificial sea salts and using 0.5 serial dilution.

    Table A-2. Preparation of Test Solutions at a Salinity of 30%o Using Natural Seawater or Artificial Sea Salts.1

Effluent Solution
1
2
3
4
5
Control
Total
Effluent Concentration
(%)
100
50
25
12.5
6.25
0.0
Solutions To Be Combined
Volume of Effluent
Solution (mL)
840
420
420
420
420


Volume of Diluent
Seawater (30%o) (mL)
 
Solution 1 + 420
Solution 2 + 420
Solution 3 + 420
Solution 4 + 420
420
2,080
^-Tiii's illustration assumes: /) the use of 5 ml of test solution in each of four replicates (total of 20 mL) for the control and five concentra-
tions of effluent, 2) an effluent dilution factor of 0.5, 3) the effluent lacks appreciable salinity, and 4) 400 mL of each test concentration is
used for chemical analysis. A sufficient initial volume (840 mL) of effluent is prepared by adjusting the salinity to 30%o. In this example, the
salinity is adjusted by adding artificial sea salts to the 100% effluent, and preparing a serial dilution using 30%o seawater (natural seawater,
HSB, or artificial seawater).  Stir solutions  I hour to ensure that the salts aVssolve. The salinity of the initial 840 mL of 100% effluent is
adjusted to 30%<> by adding 25.2 g of dry artificial sea salts (FORTY FATHOMS). Test concentrations are then made by mixing appropri-
ate volumes of salinity adjusted effluent and 30%0 salinity dilution water to provide 840 mL of solution for each concentration. IfHSB alone
(I00%o) is used to adjust the salinity of the effluent, the highest concentration of effluent that could be  tested would be 70% at 30%o salinity.
     Source: EPA, 2002a.
A-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY            Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
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Appendix  B:
Apparatus and  Equipment
       Air lines, and air stones. For aerating water containing adults, or for supplying air to test solutions with
               low DO.

       Air pump. For oil-free air supply.

       Balance. Analytical, capable of accurately weighing to 0.00001 g.

       Beakers or flasks. Six, borosilicate glass or non-toxic plasticware, 1000 ml for making test solutions.

       Centrifuge. Bench-top, slant-head, variable speed for washing eggs.

       Centrifuge tubes. Conical for washing eggs.

       Compound microscope. For examining and counting sperm cells and fertilized eggs (25X and 100X).

       Count register. 2-place for recording sperm and egg counts.

       Cylindrical glass vessel. 8-cm diameter for maintaining dispersed egg suspension.

       Dissecting microscope. For counting diluted egg stock (100X).

       Environmental chamber or equivalent facility with temperature control (20C + 1C).

       Fume hood. To protect from formaldehyde fumes.

       Glass dishes. Flat bottomed, 20-cm diameter for holding sea urchins during gamete collection.

       Hemacytometer, Neubauer. For counting sperm.

       Ice bucket. Covered for maintaining live sperm after collection until test initiation.

       Laboratory sea urchins, Arbacia punctulata, culture unit. To test effluent or receiving water toxicity,
               sufficient eggs and sperm must be available from healthy adult animals.

       Meters: pH and DO, and specific conductivity. For routine physical and chemical measurements.

       Pipets, automatic. Adjustable 1 - 100 ml.

       Pipets, serological. 1-10 mL, graduated.

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

       Pipet bulbs and filters. Propipet, or equivalent.

       Reference weights, Class S. For checking performance of balance.  Weights should bracket  the expected
               weights of materials to be weighed.

       Refractometer or other method. For determining salinity.

       Samplers. Automatic sampler, preferably with sample cooling capability, that can  collect a 24-hour
               composite sample of 5 L.
                                                                                                B-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY             Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
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       Sedgwick-Rafter counting chamber. For counting egg stock and examining fertilized eggs.

       Syringes. 1 ml, and 10 ml, with 18 gauge, blunt-tipped needles (tips cut off) for collecting sperm and
               eggs.

       Thermometers. National Bureau of Standards Certified (see EPA 2002a). Used to calibrate laboratory
               thermometers.

       Thermometers, glass or electronic, laboratory grade. For measuring water temperatures.

       Transformer, 10-12 Volt. With steel electrodes for stimulating release of eggs and sperm.

       Vacuum suction device. For washing eggs.

       Volumetric flasks and graduated cylinders. Class A, Borosilicate glass or non-toxic plastic labware,
               10 - 1000 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.

       Water purification system. Millipore Milli-Q deionized water or equivalent.
B-2

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       U.S. ENVIRONMENTAL PROTECTION AGENCY            Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                    Supplement to Training Video
if'. '>.,* **.,.


   Appendix  C:

   Reagents and Consumable Materials

          Acetic acid. 10%, reagent grade, in seawater for preparing killed sperm dilutions.

          Buffers pH 4, pH 7, and pH 10. (Or as per instructions of instrument manufacturer) for standards and
                 calibration check.

          Data sheets (one set per test). For data recording (see Appendix E).

          Effluent, receiving water, and dilution water.  Test waters, including effluent, receiving, and dilution
                 water should be analyzed to ensure its quality prior to using in tests. Dilution water containing
                 organisms that might prey upon or otherwise interfere with the test organisms should be filtered
                 through a fine mesh (with 150 urn or smaller openings).

          Food. Kelp,  Laminaria sp., or romaine lettuce for the sea urchin, Arbacia punctulata.

          Formalin. 1%, in 2 ml of seawater for preserving eggs at end of test.

          Gloves, disposable; lab coat and protective eyewear. For personal protection from contamination.

          Laboratory  quality assurance samples and standards. For calibration of the above methods.

          Markers, waterproof. For marking containers, etc.

          Parafilm. To cover tubes and vessels containing test materials.

          Reagent water Distilled or deionized water that does not contain substances which are toxic to the test
                 organisms.

          Reference toxicant solutions. Reference toxicants such as sodium chloride  (NaCI), potassium chloride
                 (KCI), cadmium chloride (CdCI2), copper sulfate (CuS04), sodium dodecyl sulfate (SDS), and
                 potassium dichromate (K2Cr207), are suitable for use  in the NPDES Program and other Agency
                 programs requiring aquatic toxicity tests.

          Saline test and dilution water. The salinity of the test water must be in the range of 20%o - 30%o. The
                 salinity should vary by no more than + 2%o among the chambers on a given day. If effluent and
                 receiving water tests are conducted concurrently, the  salinities of these tests should be similar.

                 It is important to maintain a constant salinity across all treatments during a test. It is desirable
                 to match the test salinity with that of the receiving water. Two methods are available to adjust
                 salinities  a hypersaline brine (MSB) derived from natural seawater or artificial sea salts. Both
                 are described in EPA, 2002.

          Sample containers. For sample shipment and storage.

          Sea Urchins. Arbacia punctulata, minimum of 12 of each sex.

          Scintillation vials. 20 ml, disposable; to prepare test concentrations.

          Standard salt water aquarium or Instant Ocean Aquarium. Capable of maintaining seawater at 15C,
                 with appropriate filtration and aeration system.

          Tape, colored. For labeling tubes.

                                                                                                   C-l

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     U.S. ENVIRONMENTAL PROTECTION AGENCY               Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                                  Supplement to Training Video
                                                 Intentionally Left Blank
C-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                               Sperm Cell Toxicity Tests Using the Sea Urchin (Arboc/a punctufato)
                                                                            Supplement to Training Video
Appendix D:
Summary of Test Conditions and Test Acceptability
Criteria
Summary of Test Conditions and Test Acceptability Criteria for Sea Urchin, Arbacia punctulata, Fertilization Test with Effluent and
Receiving Waters (Test Method I008.0)1
Test type
Salinity
Temperature (C)
Light quality
Light intensity
Test chamber size
Test solution volume
Number of sea urchins
Number of eggs and sperm cells per chamber
Number of replicate chambers per concentration
Dilution water
Test concentrations
Dilution factor
Test duration
Endpoint
Test acceptability criteria
Sampling requirements
Sample volume required
Static, non-renewal (required)
30%0  2%o of the selected test salinity (recommended)
20C  IC (recommended) Test temperatures must not deviate by more
than 3C during the test (i.e., max. temp - min. temp S 3C) (required)
Ambient laboratory light during test preparation (recommended)
10-20 uE/m2/s, or 50 - 100 ft-c (Ambient laboratory levels) (recommend-
ed)
Disposable (glass) liquid scintillation vials (20 mL capacity), pre-soaked in
control water (recommended)
5 mL (recommended)
Pooled eggs from 4 females and pooled sperm from 4 males per test
(recommended)
About 2,000 eggs and 5,000,000 sperm cells per vial (recommended)
4 (required minimum)
Uncontaminated source of natural seawater; deionized water mixed with
MSB or artificial sea salts (available options)
Effluents: 5 and a control (required minimum) Receiving waters: 100%
receiving water (or minimum of 5) and a control (recommended)
Effluents: ^ 0.5 (recommended)
Receiving Waters: None or > 0.5 (recommended)
1 hour and 20 minutes (required)
Fertilization of sea urchin eggs (required)
70% - 90% egg fertilization in controls (required)
For on-site tests, one sample collected at test initiation, and used within
24 hr of the time it is removed from the sampling device. For off-site tests,
holding time must not exceed 36 hr before first use for NPDES compliance
testing, (required)
1 L per test (recommended)
   ^-Source: EPA, 2002a. For the purposes of reviewing WET test data submitted under NPDES permits, each test condition
   listed above is identified as required or recommended. Additional requirements may be provided in individual permits,
   such as specifying a given test condition where several options are given in the method.
                                                                                           D-l

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     U.S. ENVIRONMENTAL PROTECTION AGENCY               Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                                                  Supplement to Training Video
                                                                                                                ,",:,, t
                                                 Intentionally Left Blank
D-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacia punctulata)
                                                                              Supplement to Training Video
Appendix E:
Data Sheets
    Figure E-l. Sperm Cell Toxicity Test, Sample Data Sheet #1
       Test ID:
       Performed By:.
      Sperm Dilutions:
      Hemacytometer Count, E:
              Sperm Concentrations
       Solution Selected for Test (>5X 107 SPM):
              Dilution:  SPM/(5 X 107) =  	
                                                     X 104 = SPM "E"
                                                   "E"X40 = A =  	  SPM
                                                   "E"X20 = B =  	  SPM
                                                    "E"X5 = D =  	  SPM
                                 	 DF
                                              ((DF) X 10) -10 =  _
                                          Final Sperm Counts =  _
Egg Dilutions:
                                            Initial Egg Count: =  _
Egg Stock Concentration = Egg Count (1 ml of 1:10 dilution) X 10:    =  _
       (Allow eggs to resettle and recount until count < 200)

Volume of SW to Add to Dilute Egg Stock to 2000/mL: Egg Count - 200:  =
Verify Final Egg Count (in 1 ml of 1:10 dilution):                    =  _
       (Count should = 100 + 20 eggs/mL)
      Test Stocks:
              Sperm Stock:
                                                                              + SW, ml
              Egg Stock:
                        Volume Added/Test Vial:
                        Volume Added/Test Vial
      Test Times:
                                            Sperm Collection:
                                              Egg Collection:
                                              Sperm Added:
                                                Eggs Added:
                                              Fixative Added:
                                              Samples Read:
                                                                 (5 X 107 SPM)
                                                                 (100 uL)
                                                                 (2000/mL)
                                                                 (ImL)
      Salinities:
                                                                                             E-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                     Sperm Cell Toxicity Tests Using the Sea Urchin (Arbacio punctulata)
                                                                                       Supplement to Training Video
    Figure E-2. Sperm Cell Toxicity Test, Sample Data Sheet #2 - Raw Data
       Test ID:
       Performed by:
Time:.
Date:
                                            Egg Counts at End of Test

Cone. (%)















Replicate 1
Total















Unfert















Replicate 1
Total















Unfert















Replicate 3
Total















Unfert















Replicate 4
Total















Unfert















       Statistical Analysis:

       Analysis of variance:

       Control:
       Different from Control (P):
       Comments:
E-2

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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WHOLE EFFLUENT TOXICITY  TRAINING VIDEO SERIES  saltwater series
         Sheepshead Minnow (Cyprinodon
         variegatus) and Inland Silverside
         (Men/d/o beryllina) Larval Survival
         and Growth Toxicity Tests
         Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave,, NW
Washington, DC 20460
EPA 833-C-09-001
March 2009

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                            NOTICE

    The revision of this guide has been funded wholly or in part by the
Environmental Protection Agency under Contract EP-C-05-063. Mention of
trade names or commercial products does not constitute endorsement or
                     recommendation for use.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
Foreword
       This supplemental guide serves as a supplement to the video "Sheepshead Minnow (Cyprinodon varie-
       gatus) and Inland Silverside (Menidia beryllina) Larval Survival and Growth Toxicity Tests" (EPA, 2009).
       The methods illustrated in the video and described in this guide support the methods published in the
       U.S. Environmental Protection Agency's (EPA's) Short-term Methods for Estimating the Chronic Toxicity of
       Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA, 2002a), referred
       to as the Saltwater Chronic Methods Manual. The video and this guide provide details on preparing for
       and conducting the test based on the expertise of personnel at the following EPA Office of Research and
       Development (ORD) laboratories:

           National Health and Environmental Effects Research Laboratory (NHEERL) - Atlantic Ecology Division
           in Narragansett, Rhode Island

           NHEERL - Gulf Ecology Division in Gulf Breeze, Florida

           National Exposure Research Lab (NERL) - Ecological Exposure Research Division (EERD) in
           Cincinnati, Ohio

       This guide and its accompanying video are part of a series of training videos produced by EPA's Office of
       Wastewater Management. This Saltwater Series includes the following videos and  guides:

           "Mysid (/Amen'camys/s bahia) Survival, Growth, and Fecundity Toxicity Tests"

           "Culturing/Americamys/s bahia"

           "Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia punctulata"

           "Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests"

           "Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
               and Growth Toxicity Tests"

       The Freshwater Series, released in 2006, includes  the following videos and guides:

           "Ceriodaphnia Survival and Reproduction Toxicity Tests"

           "Culturing of Fathead Minnows (Pimephales promelas)"

           "Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests"

       All of these videos are available through the National Service Center for Environmental Publications
       (NSCEP) at 800 490-9198 or nscep@bps-lmit.com.

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U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon vor/egotus) and Inland Silverside (Men/did beryllina)
                                                          Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                                                                                :  .>-* v-**'S'-;,-<*r
                                                Intentionally Left Blank

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    U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegotus) and Inland Silverside (Menidia beryffina)
                                               Larval Survival and Growth Toxicity Test  Supplement to Training Video
Contents
      Foreword	i
      Introduction	1
      Background	1
      Care and Feeding of Adults and Larvae	1
      Culture Water	2
      Photoperiod	'.	2
      Culture Vessels	2
      Water Delivery Systems	2
      Food Preparation	2
      Obtaining Larvae for Toxicity Tests	3
      Culture Water	4
      Photoperiod	5
      Culture Vessels	5
      Water Delivery Systems	5
      Food Preparation	5
      Obtaining Larvae for Toxicity Tests	5
      Test Method	6
      Effluent Sampling	6
      Dilution Preparation	6
      Routine Chemistries	,	7
      Renewals	7
      Feeding	8
      Test Termination	8
      Test Acceptability and Data Review	9
      Other Procedural Considerations	9
      Citations and Recommended References	9
      Glossary	 Glossary-1
      Appendix A: Preparing Hypersaline Brine (HSB)	A-l
      Appendix B: Preparing Brine Shrimp and  Rotifers for Feeding	B-l
      Appendix C: Apparatus and Equipment - Sheepshead Minnow and Inland Silverside Tests....  C-l
      Appendix D: Reagents and Consumable Materials	D-l

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U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina)
                                           Larval Survival and Growth Toxicity Test  Supplement to Training Video
  Appendix E: Summary of Test Conditions and Test Acceptability Criteria	E-l

  Appendix F: Data Sheets	F-l

  FIGURES
  Figure 1. Embryonic development of sheepshead minnow, Cyprinodon variegatus: A. Mature
  unfertilized egg, showing attachment filaments and micropyle, X33; B. Blastodisc fully devel-
  oped; C/D. Blastodisc, 8 cells; E. Blastoderm, 16 cells; F. Blastoderm, late cleavage stage; G.
  Blastoderm with germ ring formed, embryonic shield developing; H. Blastoderm covers over % of
  yolk, yolk noticeably constricted; I. Early embryo. (Continued, J - 0 on page 4)	3

  Figure 1 (continued). Embryonic development of sheepshead minnow, Cyprinodon variegatus: J.
  Embryo 48 h after fertilization, no segmented throughout, pigment on yolk sac and body, otoliths
  formed; K. Posterior portion of embryo free from yolk and moves freely within egg membrane, 72
  h after fertilization; L. Newly hatched fish, actual length 4 mm; M. Larval fish 5 days after hatch-
  ing, actual length 5 mm; N. Young fish 9 mm  in length; 0. Young fish  12  mm in length	4

  Figure 2. Inland silverside, Menidia beryllina: A. Adult, ca. 64 mm SL; B. Egg (diagrammatic), only
  bases of filaments shown; C. Egg, 2-cell stage; D. Egg, morula stage; E.  Advanced embryo, 2Vz
  days after fertilization	6

  Figure 3. Glass test chamber with sump area. Modified from Norberg and  Mount (1985)	7

  Figure C-l. Glass test chamber with sump area. Modified from Norberg and Mount (1985). ... C-2

  Figure F-l. Data Form for the Sheepshead Minnow and Inland Silverside, Larval Survival and
  Growth Toxicity Test. Daily Record of Larval Survival and Test Conditions	F-l

  Figure F-2. Data Form for the Sheepshead Minnow and  Inland Silverside,  Larval Survival and
  Growth Toxicity Test. Summary of Test Results	F-3

  Figure F-3. Data Form for the Sheepshead Minnow and  Inland Silverside,  Larval Survival and
  Growth Toxicity Test. Dry Weights of Larvae	.. F-4

  TABLES
  Table A-l. Preparation of Test Solutions at a Salinity of 20%0 Using MSB for a Final Test
  Concentration Volume of 4000 mL	A-2

  Table E-l. Summary of Test Conditions and Test Acceptability Criteria for the Sheepshead
  Minnow, Cyprinodon variegatus, Larval Survival and Growth Test with Effluents and Receiving
  Waters (Test Method 1004.0)	E-l

  Table E-2. Summary of Test Conditions and Test Acceptability Criteria for the Inland Silverside,
  Menidia beryllina, Larval Survival and Growth Test with Effluents and Receiving Waters (Test
  Method 1006.0)	E-2

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     U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegotus) and Inland Silverside (Menidia beryllina)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
Introduction

       This guide accompanies the Environmental Protection Agency's (EPA's) video training for conducting
       sheepshead minnow (Cyprinodon variegatus) and inland silverside (Menidia beryllina) larval survival and
       growth toxicity tests (EPA, 2009). The test methods are found in Short-term Methods for Estimating the
       Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA,
       2002a). The tests were developed by EPA's Office of Research and Development's (ORD's) National Health
       and Environmental Effects Research Laboratory - Aquatic Ecology Division (NHEERL-AED) in Narragansett,
       Rhode Island. The material presented in both the video and this guide summarizes the methods but does
       not replace a thorough review and understanding of the methods by laboratory personnel before conduct-
       ing the test.
Background
       Under the National Pollutant Discharge Elimination System (NPDES) program (Section 402 of the Clean
       Water Act), EPA uses toxicity tests to monitor and evaluate effluents for their toxicity to biota and their
       impact on receiving waters. By determining acceptable or safe concentrations for toxicants discharged
       into receiving waters, EPA can establish NPDES permit limitations for toxicity. These whole effluent toxicity
       (WET) permit limitations regulate pollutant discharges on a whole effluent effect basis rather than solely by
       a chemical specific approach.

       Whole effluent toxicity methods measure the synergistic, antagonistic, and additive effects of all the chemi-
       cal, physical, and additive components of an effluent that adversely affect the physiological and biochemi-
       cal functions of the test organisms. Therefore, healthy organisms and correct laboratory procedures are
       essential for valid test results. Laboratory personnel should be very familiar with the test methods and with
       sheepshead minnows and inland silverside handling techniques before conducting a test.

       This supplemental guide covers the procedures for conducting the test according to EPA's promulgated
       methods (40 CFR Part 136; EPA, 2002c) and also provides some helpful information that is not presented
       in the Saltwater Chronic Methods Manual (EPA, 2002a).

       This guide summarizes methods developed at ORD for measuring effects on larval survival and growth of
       the sheepshead minnow Cyprinodon variegatus and the inland silverside Menidia beryllina after exposure
       to complex effluents in marine or estuarine environments. These short-term tests span an exposure time
       of 7 days to estimate the chronic toxicity of effluent or receiving water on newly-hatched larvae in a static
       renewal exposure system. The methods described in this guide and demonstrated in the accompanying
       video are detailed in the EPA methods manual, Short-term Tests for Estimating the Chronic Toxicity of
       Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA, 20023)1.

Care and Feeding of Adults and  Larvae

       SHEEPSHEAD MINNOWS
       Adult sheepshead minnows (Cyprinodon variegatus)  can be field collected from Atlantic and Gulf of Mexico
       coastalestuaries south of Cape Cod using near-shore nets, purchased from commercial biological supply
       houses, or raised from young fish to maturity in the laboratory. To minimize inbreeding, use of feral brood
       stocks or first generation laboratory fish is recommended. Fish that are field-caught should be held for a
       minimum of 2 weeks before use in testing to determine that they are healthy and not injured.
1 The methods for these two species are presented together in the video and this guide because they are conducted in a very similar man-
ner. The complete methods in the Saltwater Chronic Methods Manual are presented in Section  II (Sheepshead Minnows) and Section 13
(Inland Silverside).
                                                                                                     I

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U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Men/did beryllina)
                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                                                     -  >u - ;,ft, s*  r f-   . j-

   CULTURE WATER
   The quality of water used for test organism culturing and for dilution water in toxicity tests is extremely
   important. Water for these two uses should come from the same source. Holding and rearing tanks and any
   area used for manipulating live sheepshead minnows should be located in a room or space separated from
   that in which toxicity test are to be conducted.

   The salinity of the culture systems should between 20%o and 30%o. Water temperature for the brood stock
   should be maintained at 24C - 26C. The holding and rearing tanks should be aerated so that the dis-
   solved oxygen is not less than 4.0  ppm.

   Replace approximately 10% of the culture water every 2 weeks, or 25% monthly. The culture water should
   be clear. If the water appears cloudy of discolored, replace at least 50% of it.  Replacement water should be
   well oxygenated and at the same temperature and salinity as the existing culture water. Salinity is main-
   tained at the proper level by adding deionized water to compensate for evaporation. Artificial seawater is
   prepared by dissolving artificial sea salts in deionized water to a salinity of 20%o - 30%o (see Appendix A
   for preparation of hypersaline brine solution [MSB]).

   PHOTOPERIOD
   The culture conditions should  include a photoperiod of 16 hours light and 8 hours dark (EPA, 2002a). The
   light quality and intensity should be at ambient laboratory levels, which is approximately 10 - 20 uE/m2/s
   or 50 to 100 foot candles (ft-c) (EPA, 2002a).

   CULTURE VESSELS
   Holding tanks are kept at ambient laboratory temperature (25C) until the fish reach sexual maturity (3-5
   months post hatch) at which time they can be used for spawning. Mature sheepshead minnows have an
   average length of approximately 27 mm  for females and 34 mm for males. Once mature, males will begin
   to exhibit sexual dimorphism and initiate territorial behavior. Once sexually mature, hold the adults in water
   reduced to 18C - 20C.

   To avoid excessive build  up of algal growth, periodically scrape the walls of the culture system. Some of the
   algae will serve as a supplement to the diet of the fish'. A partial activated carbon "charcoal" change in the
   filtration systems should be done monthly or as needed. The detritus (dead brine shrimp nauplii and cysts,
   adult brine shrimp, other organic material accumulation) should be siphoned from the bottom of rearing
   and holding aquaria or tanks each week or as needed.

   WATER DELIVERY SYSTEMS
   Adult sheepshead minnows (>1 month) are kept in natural or artificial seawater in  a flow-through or recircu-
   lating aerated glass aquarium that is equipped with an undergravel or outside biological filter, or cartridge
   filter. Static systems are  equipped with an undergravel filter. Recirculating systems are equipped with an
   outside biological filter constructed in the laboratory using a reservoir system of crushed coral, crushed
   oyster shells or dolomite and gravel, charcoal, floss, or a commercially available cartridge filter or an
   equivalent system.

   FOOD PREPARATION
   The adult sheepshead minnows are fed flake food three to four times daily, supplemented with frozen adult
   brine shrimp.

   The larvae are fed newly hatched Artemia nauplii and crushed flake food, ad libitum, daily. The Artemia
   should be cultured in the laboratory in order to provide 24 - 48 hour old nauplii. Appendix B describes in
   detail how to culture Artemia.

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     U.S. ENVIRONMENTAL PROTECTION AGENCY
                                           Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Men/did beryffina)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                       Figure 1. Embryonic development of Sheepshead minnow, Cyprinodon
                                       variegatus: A. Mature unfertilized egg, showing attachment filaments
                                       and micropyle, X33; B. Blastodisc fully developed; C/D. Blastodisc, 8
                                       cells; E. Blastoderm, 16 cells; F. Blastoderm, late cleavage stage; G.
                                       Blastoderm with germ ring formed, embryonic shield developing; H.
                                       Blastoderm covers over 3A of yolk, yolk noticeably constricted; I. Early
                                       embryo. (Continued, J - 0 on page 4).
OBTAINING LARVAE FOR
TOXICITY TESTS
For the Sheepshead larval
survival and growth toxicity
test, larvae that are less than
24 hours old are needed at the
start of the test. To have the
appropriate age larvae at the
start of a test, induce the min-
nows to spawn by raising the
To keep the egg collecting
screens clean, feed the
spawning fish while the
collecting screen is removed
for egg collection.
       system temperature to 25C
       approximately 1 week before
       the start of the test. This
       gradual temperature increase
       is started in the morning. By
       afternoon, transfer the adults
       (at least five females and three
       males) to a spawning chamber,
       or basket made from 3-5
       mm NITEX screen, within an
       aquarium outfitted with a mesh
       screen (150 - 250 urn mesh)
       under the basket or on the
       bottom. The fish will begin to
       spawn within  24 hours and the eggs will fall through the basket onto the mesh collecting
                                        G                     H
                                Source: Kuntz, 1916 in EPA, 2002a.
                                                                              screen.
       Collect eggs daily by washing the eggs off of the screen into a large tray. Roll the eggs gently on the screen
       during collection, pressing any food or waste through, leaving the eggs on top of the screen. Embryos will
       tend to stick together due to the presence of adhesive threads. After embryos have been manipulated,
       wash them by placing them in a 250-um sieve and rinsing them with seawaterfrom a squeeze bottle. This
       should reduce any fungal contamination of the embryos.

       Females also can be induced to spawn artificially by intraperitioneal injection with human chorionic gonad-
       otrophin (HCG) hormone. Natural spawning is preferable because repeated spawnings can be obtained
       from the same brood stock. Additional details on forced spawning are provided in section 11.6.15 of the
       Saltwater Chronic Methods Manual (EPA,  2002a).

       The collected embryos should be checked under a dissecting microscope to identify any abnormal or unfer-
       tilized eggs. The embryos should be in stages C - G as illustrated in Figure 1.

       After collection, incubate the collected minnow embryos in seawater at 25C, 20%o - 30%o salinity, and
       16-hour light and 8-hour dark photoperiod  for 5 - 6 days with aeration and daily water changes.

       At 48 hours after collection, check the embryos under a dissecting microscope and discard any abnormal or
       unfertilized eggs. At this time, the embryos should be at stages I or J as illustrated in  Figure 1. To conduct one

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                      Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina)
                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
  test with four replicates of
  15 larvae and five efflu-
  ent concentrations plus
  a control, collect approxi-
  mately 400 viable embryos
  for incubation at this stage.
  Reducing the salinity, raising
  the temperature, or chang-
  ing the water can help
  induce hatching. If culture
  dishes are used, they should
  be covered to reduce evapo-
  ration which could increase
  salinity.

  For the sheepshead minnow
  growth and survival toxicity
  test, use larvae that hatch
  less than 24 hours before
  the start of the test. If some
  embryos hatch earlier than
  24 hours prior to the test
  start, remove them but keep
  them to supplement the
  younger larvae in case there
  are not be enough larvae at
  the start of the test. If this is
  done, larvae should not be
  more than 48 hours old and
  should all be within 24 hours
  of the same age. Selection
  of the older larvae should be
  randomized by placing them
  back  into the pool before
  selection.

  INLAND SILVERSIDE
Figure 1 (continued).  Embryonic development of sheepshead
minnow, Cyprinodon variegatus: J. Embryo 48 h after fertilization, no
segmented throughout, pigment on yolk sac and body, otoliths formed;
K. Posterior portion of embryo free from yolk and moves freely within
egg membrane, 72 h after fertilization; L. Newly hatched fish, actual
length 4 mm; M. Larval fish 5 days after hatching, actual length 5 mm;
N. Young fish 9 mm in length; 0. Young fish 12 mm in length.
From Kuntz, 1916 in EPA, 2002a.
   Inland silversides (Menidia beryllina) also can be obtained by beach seine from Atlantic and Gulf of Mexico
   coastal estuaries, from biological supply houses, or by raising young fish in the laboratory. Gravid females
   can be found in low salinity waters along the Atlantic coast during April to July. If beach seines (3 mm - 6
   mm mesh) are used, silversides should not be landed onto the beach as they are very sensitive to  handling
   and should not be removed from water by net  only by bucket or beaker. Several species of silversides
   may be included in field caught specimens (e.g., M. beryllina, M. menidia, and M. peninsulas); care should
   be taken to identify and separate the species.

   If fish are collected from the field, record the temperature and salinity at each collection site so that the
   conditions can be maintained in the culture tanks. After transfer to laboratory culture tanks, slowly intro-
   duce laboratory water (maximum  change of 2C/day and 5%o salinity/day) to bring the water up to 25C and
   20%o - 32%o.

   CULTURE WATER
   Only natural seawater is recommended for the culture and maintenance of the more sensitive silverside
   brood stock. Maintain holding and spawning tanks at a temperature of 25C and a salinity of 20%o - 32%o.

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U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryl/ina)
                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
   PHOTOPERIOD
   The culture conditions should include a photoperiod of 16 hours light and 8 hours dark (EPA, 2002a). The
   light quality and intensity should be at ambient laboratory levels, which is approximately 10 - 20 uE/m2/s
   or 50 - 100 foot candles (ft-c).

   CULTURE VESSELS
   Adult inland silverside should be stocked in tanks of a minimum volume of 150L at a density of 50 fish/
   tank. Detritus should be siphoned off from the bottom weekly, or as needed.

   WATER DELIVERY SYSTEMS
   Adult inland silversides are kept in a flow-through or recirculating aerated glass aquarium that is equipped
   with an undergravel or outside biological filter, or cartridge filter. Static systems are equipped with an
   undergravel filter. Recirculating systems are equipped with an outside biological filter constructed in the
   laboratory using a reservoir system  of crushed coral, crushed oyster shells or dolomite and gravel, char-
   coal, floss, or a commercially available cartridge filter or an equivalent system.

   FOOD  PREPARATION
   Feed silverside larvae the rotifer Brachionus plicatilis until 4-6 days post-hatch, and the smallestX\rtem/a
   nauplii available (<12 hour old) beginning on day 5. After day 7, feed the larvae with Artemia only and
   increase the size to 12 - 24 hours old. Food preparation instructions are  provided in Appendix B.

   The adult inland silversides should be fed flake food or frozen brine shrimp twice daily and Artemia nauplii
   once daily.

   The larvae are fed newly hatched Artemia nauplii and crushed flake food, ad libitum, daily. The Artemia
   should be cultured in the laboratory in order to provide 24 - 48 hour old nauplii. Appendix B describes in
   detail how to culture Artemia.

   OBTAINING LARVAE FOR TOXICITY TESTS
   Inland silversides are sexually mature after 1-2 months. In the wild, eggs are adhered to submerged
   vegetation. In the laboratory, silversides are encouraged to spawn by placing polyester aquarium filter fiber
   in the tanks. The fiber (~ 15 cm x 10 cm x 10 cm) is suspended on a string 8 cm - 10 cm below the surface
   of the water and in contact with the side of the tank. These should be placed into the tank 14 days prior to
   the beginning of a test. Place the floss directly above an airstone to keep  it aerated, and weigh it down to
   keep it from floating on the surface.

   When the fish spawn into the fiber, the hard, light yellow embryos (-0.75 mm in diameter) can be separated
   from the fibers by hand, or the eggs and fiber can be placed together into a 10-gallon aquarium. The floss
   should be suspended 8 cm  - 10 cm below the surface of the water and should be stretched to keep the
   embryos from being-crowded. Lightly aerate the tank and hold the temperature at 25C.

   Larvae will hatch in  6 - 7 days when incubated at 25C and  maintained in seawater ranging from 5%o -
   30%0. The larvae will free themselves from the fibers at which time they are easily identified and should be
   removed. The newly hatched larvae will range from  3.5 mm - 4.0 mm in total length. Figure 2 illustrates
   the life stages of the inland  silverside.

   For the inland silverside larval survival and growth toxicity test, use 7- to 11-day-old larvae. For one test
   using 15 larvae for each of four replicates and five test concentrations plus a control, approximately 400
   larvae are needed.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                           Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia faery/lino)
                                                   Larval Survival and Growth Toxicity Test  Supplement to Training Video
Test  Method

       EFFLUENT SAMPLING
       For both species, handle efflu-
       ent and receiving water sam-
       ples in the same manner. Store
       effluent or receiving waters in
       an incubator or refrigerator at
       0C - 6C until the tests begin,
       but not longer than 36 hours
       if being used for compliance
       fora NPDES permit. Prepare
       dilutions of the effluent sample
       using a 0.5 dilution factor (e.g.,
       6.25%, 12.5%. 25%, 50%, and
       100%). If a  high rate of mortal-
       ity is observed during the first
       1-2 hours, additional repli-
       cates in the lower ranges of
       effluent concentration should
       be added.

       The tests require about 5-6
       L of each effluent or receiv-
       ing water sample each day,
       enough for renewing four
       replicates of each concentra-
       tion plus the control and for
       performing chemical analyses.

       It is essential to maintain
       constant salinity among
       treatments and treatment
       replicates throughout the
       test. Use concentrated sea-
       water or hypersaline brine
       (MSB) to keep the salinity
       of the solutions between
       20%o and 30%o for the
       sheepshead minnows, and
       between 5%o and 30%o for
       the inland silversides.  Before
       adding the solutions to the
       test chambers, wartn the
       samples to 25C in a water
       bath.  Keep the temperature
       constant (25C + 1C) for the
       duration of the test.

       DILUTION PREPARATION
  Figure 2. Inland silverside, Menidia berylllna: A. Adult, ca. 64 mm SL;
  B. Egg (diagrammatic), only bases of filaments shown; C. Egg, 2-cell
  stage; D. Egg, morula stage; E. Advanced embryo, 2V2 days after
  fertilization..
                                     G Larva 8.9 mm TL
     D              E

  From Martin and Drewry, (978 in EPA, 2002a.
Dilution Water
The type of dilution water used to make the test concentrations is
dependent on the objectives of the test. Any specific requirements
included in NPDES permits should be followed.  The Saltwater Chronic
Methods Manual (Section 7) provides the following guidelines:
  If the test is conducted to estimate the absolute chronic toxicity
of the effluent, synthetic dilution water should be used. If the cultures
were maintained in different water than used for dilution water, a
second set of control replicates should be conducted using the culture
water.
  If the test is conducted to estimate the chronic toxicity of the
effluent in uncontaminated receiving waters, the test can be
conducted using a grab sample of the receiving waters collected outside
the influence of the outfall, other uncontaminated waters, or standard
dilution water with the same salinity as  the receiving waters. If the
cultures were maintained in different water than used for dilution water,
a second set of control replicates should be conducted using the culture
water.
  If the test is conducted to estimate the additive or mitigating
effects of the effluent on already  contaminated receiving
waters, the test must be conducted using receiving waters collected
outside the influence of the outfall. Controls should be conducted using
both receiving water and culture water.
       Set out the test chambers.
       Typically, there will be at least five dilutions plus one control, and a minimum of four replicates. For both
       species NHEERL-AED uses glass chambers equipped with a screened-off sump area (see Figure 3). One

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                      Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Menidia bery/Jina)
                                             Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                Figure 3. Glass test chamber with sump area. Modified from Norberg
                                and Mount (1985).
                                                                                Glass
                                                                             Reinforcements
                                                                                             Sump
                                    Source: EPA, 2002a.
thousand ml glass or
disposable plastic beakers
also can be used as test
chambers. Add a small
amount of clean seawater
to each chamber, enough to
cover the bottom to a depth
of about 1cm.
Pipet two or three larvae at
a time into each chamber,
adding larvae to all cham-
bers; then start again, add-
ing more until each cham-
ber contains the required
number of larvae  a
minimum of 10. Use  a mini-
mum amount of seawater to
deposit the animals into the
containers to avoid diluting the effluent samples further. Using a white background or a light table facili-
tates counting the larvae in the chambers. Since clean seawater is in all of the chambers, larvae can be
exchanged among test chambers until all contain the correct number. Because the inland silverside larvae
are sensitive to handling, it may be best to distribute them into chambers containing control solution 1 day
before the start of the exposure period.

Randomly apply colored labels to the chambers to indicate treatment and replicates.  Fill each chamber
with  approximately 750 ml of the appropriate test solution, pouring through the sump area or down the side.
Each test chamber should contain a minimum of 50 ml of test solution/larvae and a depth of at least 5 cm.

ROUTINE CHEMISTRIES
           Measure the initial temperature, salinity, and dissolved oxygen (DO) in each chamber. Record
           all measurements on the test data sheet. Copies of the data sheets used at NHEERL-AED are
            provided in Appendix F.

             When  all measurements have been taken and recorded, place the chambers in a 25C
             water bath according to a random numbers table. Keep the chambers in those same posi-
           tions for the duration of the test.

RENEWALS
Each day, the test and control solutions must be replenished. Prepare new dilutions daily from effluent
stored at 0 - 6C. When tests are performed on site, effluent and receiving water should be collected
daily. Off-site toxicity tests are often performed with effluent collected on days 1, 3, and 5 of the exposure
period. Again, do not store the effluent samples longer than 36 hours before use. Warm the solutions to
25C in a water bath just before adding to the chambers.

Temperature and salinity should be maintained  under carefully controlled conditions across all test
concentrations.and replicates throughout the test. Each day before changing the solutions, measure and
record the temperature in each chamber. Maintain the chambers at 25C + 1C, and supply 16 hours of
ambient laboratory light and 8 hours of darkness each day for both species. Measure and record the salin-
ity from each chamber every day as well, before renewing the test solutions. Note that there should be no
more than a 2%o salinity difference between any two chambers on a given day. If receiving water and efflu-
ent tests are conducted concurrently, the effluent salinity should be adjusted to match the receiving water

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U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Men/did beryl/ino)
                                             Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                                                 -   --  - h .".  -  - -..V*! V3*4

  sample if possible. Monitor DO concentrations each day and record the data on the data sheet. If DO falls
  below 40% saturation in any one of the exposure chambers, all chambers must be aerated.

  Before changing the test solutions, count and record the number of live larvae in each replicate, discard-
  ing any dead animals. Then remove any uneaten Artemia from the chamber using a siphon or a large pipet.
  To avoid'removing test animals along with uneaten food, set the chambers on a light box or light table to
  better observe the larvae. Besides making the larvae more visible, the light also serves to concentrate the
  nauplii on the bottom of the chamber. Siphon the water and remaining Artemia into a large beaker or white
  plastic tray. Individual larvae that are accidentally removed can be seen easily in the beaker, and should be
  returned to their respective test chambers. Note the accidental siphoning of any larvae in the test records.
  Once the solution in the test chamber is emptied to a depth of 7 - 10 mm, slowly and carefully add approxi-
  mately 500 - 750 mL of new test solution, pouring down the side of the chamber or into the sump area to
  avoid excessive turbulence. After changing all the solutions, return the chambers to their same randomized
  positions in the water bath and feed the  larvae.

  FEEDING
  Proven quality/Artem/a nauplii should be used to feed the larvae daily throughout the test. Two concentra-
  tions of prepared nauplii are used sequentially during the exposure period. Detailed instructions for cultur-
  ing Artemia are included in Appendix B. The first food solution used for day 0 -  2 consists of 4 mL concen-
  trated Artemia nauplii in 80 mL seawater. Feed each replicate 2 mL of this solution on the first 2 days of
  the test. The 2 mL volume should yield approximately 0.10 g wet weight of Artemia nauplii. Care should be
  taken to swirl the solution to maintain a constant distribution of Artemia and each 2 mL  portion should be
  drawn  individually to avoid differences in feeding rates due to the settling of Artemia in the dropper.

  For days 3 - 6 of the test, feed the larvae  2 mL per replicate of a more concentrated solution of 6 mL of
  concentrated Artemia in 80 mL of seawater. This 2 mL volume should yield approximately 0.15 g wet weight
  Artemia nauplii. Uneaten Artemia should be siphoned out of the chambers each day so that the larvae eat
  newly hatched Artemia and to avoid depletion of DO within the chamber. On day 7, the larvae are not fed.

  It is important that all chambers receive the same amount of food throughout the test. If the survival rate in
  any chamber falls below 50%, reduce the amount of food supplied to that chamber by Vb for the remainder
  of the test. Cover the chambers between feedings to reduce evaporation.

  TEST TERMINATION
  At the end of the test, on day 7, the larvae are counted to determine survival rate. Working with groups of
  replicates,  remove any dead larvae from the chambers, carefully recording the number of surviving ani-
  mals.  Record the final temperature, salinity, and DO for each chamber.

  Pour the contents of each chamber through a 500-um mesh screen over a large beaker. Quickly submerge
  the screen  in an ice and deionized water bath. The cold will immobilize the fish, and swirling the screen in
  the deionized water will wash away uneaten Artemia and salts that may interfere with the weight determi-
  nation. Dry the animals for immediate weighing or preserve them for later drying in separate scintillation
  vials containing 4% formalin or 70% ethanol. To dry the surviving animals, place all  of the fish from each
  replicate into a labeled, pre-weighed aluminum weighing boat, and dry the fish at 60C for 24 hours, or
  at 105C for 6 hours. Gloves should be worn or forceps should be used to handle the aluminum weighing
  boats because oil from  skin could affect weight differences.

  After drying, and until they are weighed, place the dried larvae directly into a desicatorto prevent moisture
  from the air adsorbing to the samples. Weigh each sample to the nearest 0.01 mg.  Because small differ-
  ences  in weight or appearance can easily change the test results, it is critical to record observations and
  measurements clearly and accurately. Determine the weight of the larvae alone by subtracting the weight
  of the weigh boat. Divide the final dry weight by the number of larvae in the sample to determine the aver-

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    U'S' ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina)
                                                 Larval Survival and Growth Toxicity Test  Supplement to Training Video
       age dry weight of the surviving larvae. This average weight is then compared statistically to the control
       animals' average weight to identify any effluent effects on the fishes' growth.

       TEST ACCEPTABILITY AND DATA REVIEW
       Test data are reviewed to verify that EPA's WET test methods' test acceptability criteria (TAG) requirements
       fora valid test have been met. For the test to be considered acceptable, control survival must be > 80% for
       both species. The average dry weight of unpreserved control larvae must be > 0.60 mgforthesheepshead
       minnow, and > 0.50 mgforthe inland silverside. Minimum dry weights for preserved animals are > 0.50 mg
       for the sheepshead minnow and > 0.43 mg for the inland silverside.

       The concentration-response relationship generated for each multi-concentration test must be  reviewed to
       ensure that calculated test results are interpreted appropriately. In conjunction with this requirement, EPA
       has provided recommended guidance for concentration-response relationship review (EPA, 2000a).

       EPA's promulgated toxicity testing method manuals (EPA, 2002a, b) recommend the use of point estima-
       tion technique approaches for calculating endpoints for effluent toxicity tests under the NPDES program.
       The promulgated methods also require a data review of toxicity data and concentration-response data, and
       require calculating the percent minimum significant difference (PMSD) when point estimation  (e.g., LC50,
       IC25) analyses are not used. EPA specifies the PMSD must be calculated when NPDES permits require sub-
       lethal  hypothesis testing. EPA also requires that variability criteria be applied as a test review step when
       NPDES permits require sub-lethal hypothesis testing endpoints (i.e., no observed effect concentration
       [NOEC] or lowest observed effect concentration [LOEC]) and the effluent has been determined to have no
       toxicity at the permitted receiving water concentration (EPA, 2002b). This reduces the within-test variabil-
       ity and increases statistical sensitivity when test endpoints  are expressed using hypothesis testing rather
       than the preferred point estimation techniques.

       OTHER PROCEDURAL CONSIDERATIONS
              Keep careful records throughout the test.

              Record any deaths and  whether any larvae were accidentally siphoned out of their chamber.

              Take special note  of any behavioral changes that the larvae may exhibit, or any physical
               abnormalities.

              Note the results of the chemical and physical measurements taken during the test.

       These data should be carefully compiled and are considered important clues to how the effluent may affect
       marine animals. The methods manual, Short-term Methods  for Estimating Chronic Toxicity of Effluents and
       Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA, 2002a) details the procedure for
       data analysis.

       The larval survival and growth toxicity tests described  here are currently used to assess the potential
       toxic effects of complex chemical mixtures on marine and estuarine organisms. Used in conjunction with
       chemical-specific methods, these tests can provide a comprehensive and  effective approach to assessing
       the impact of complex effluents discharged to marine and estuarine environments.


Citations and  Recommended  References

       Gripe, G.M., B.L. Hemmer, L.R. Goodman, and J.C.Vennari. 2009. Development of a Methodology for
               Successful Multigeneration Life-Cycle Testing of the Estuarine Sheepshead Minnow, Cyprinodon
             i  variegatus. Archives of Environmental Contamination and Toxicology. April 56(3):500-8.

       EPA.  1985. Aquatic Toxicity Testing Seminar Manual.  National Health and Environmental Effects Research
               Laboratory - Aquatic Ecology Division, Narragansett, Rl.  NHEERL-AED Contribution No. 796.

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    UiS' ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidio beryllina)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                                                  '  <'..     -'*

       EPA. 1987a.  Guidance manual for rapid chronic toxicity tests on effluents and receiving waters with
               larval inland silversides (Menidia beryllina). Contribution No. 792. Heber,  M.A., M.M. Hughes,
               S.C. Schimmel, and D.A. Bengston. In:  Schimmel, S.C. ed., Users guide to the conduct and
               interpretation of complex effluent toxicity tests at estuarine/marine sites. Environmental Research
               Laboratory, U.S. EPA, Narragansett, Rl  02882. Contribution No. 796, 265 pp.

       EPA. 1987b.  Guidance manual for conducting complex effluent and receiving water larval fish growth-survival
               studies with the sheepshead minnow (Cyprinodon variegatus).  Contribution No. x!04. Hughes, M.M.,
               M.A.  Heber, S.C. Schimmel, and WJ. Berry. In: Schimmel, S.C. ed., Users guide to the conduct and
               interpretation of complex effluent toxicity tests at estuarine/marine sites. Environmental Research
               Laboratory, U.S. EPA, Narragansett, Rl 02882.  Contribution No. 796, 265 pp.

       EPA. 1989.  Biomonitoring for Control of Toxicity in Effluent Discharges to the Marine Environment.
               U.S. EPA Center for Environmental Research Information, Cincinnati, OH. U.S. EPA National Health
               and Environmental Effects Research Laboratory - Aquatic Ecology Division, Narragansett, Rl.
               EPA/625/8-89/015.

       EPA. 1991.  Technical Support Document for Water Quality-based Toxics Control. U.S. EPA Office of Water
               Enforcement and Permits, Washington, D.C. EPA-505-2-90-001.

       EPA. 2000a. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR
               136). Office of Water, Washington, D.C. EPA 821-B-00-004.

       EPA. 2000b. Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications
               Under the  National Pollutant Discharge Elimination System Program. Office of Wastewater
               Management, Washington, D.C. EPA 833-R-00-003.

       EPA. 2002a. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
               Waters to Marine and Estuarine Organisms, Third Edition (Saitwater Chronic Methods Manual).
               Environmental Protection Agency, Washington, DC. EPA-821-R-02-014.

       EPA. 2002b. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
               and Marine Organisms, Fifth Edition. (Acute Methods Manual).  Office of Water, Cincinnati, OH.
               EPA-821-R-02-012.

       EPA. 2002c. Final Rule. 40 CFR Part 136. Guidelines Establishing Test Procedures for the Analysis of
               Pollutants; Whole Effluent Toxicity Test Methods. 67 FR 69952-69972, November 19, 2002.

       EPA. 2009.  Sheepshead Minnow (Cyprinodon  variegatus) and Inland Silverside (Menidia beryllina) Larval
               Survival and Growth Toxicity Test. Supplement to Training Video. Whole Effluent Toxicity Training
               Video-Saltwater Series. March 2009.  EPA 833-C-09-001

       Johns, D.M., WJ. Berry, and W. Walton.  1981.  International Study on Artemia, XVI. Survival, growth, and
               reproduction potential of the mysid, Mys/dops/s bah/a  Molenock fed various geographical  collec-
               tions of the brine shrimp, Artemia. J. Exp. Mar. Biol. Ecol. Vol. 53,  pp. 209-219.

       Klein-MacPhee, G., W.H. Howell, and A.D.  Beck. 1982. International Study on Artemia, XX.
               Comparison of a reference and four geographical strains of Artemia as food for winter flounder
               (Pseudopleuronectes americanus) larvae. Aquaculture.  Vol. 29, pp. 279-288.

       Leger, P., and P. Sorgeloos. 1984. International Study on Artemia, XXIX. Nutritional value of Artemia nau-
               plii from various geographical origins for the mysid, Mysidopsis bahia (Molenock). Mar. Ecol. Prog.
               Ser. Vol. 15, pp. 307-309.
10

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U'S- ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon vor/egatus) and Inland Silverside (Men/did beryllina)
                                               Larval Survival and Growth Toxicity Test  Supplement to Training Video
   Norberg, TJ. and DJ. Mount. 1985. A new fathead minnow (Pimephales promelas) Subchronic toxicity
           test. Environ. Toxicol. Chem. 4(5):711-718.

   Persoone, G., P. Sorgeloos, 0. Roels, and E. Jaspers eds. 1980. The brine shrimp Artemia. Vols. 1-3.
           Proceedings of the International Symposium on the brine shrimp Artemia satina, Corpus Christi,
           Texas. 1979. Universal Press, Wetteren, Belgium.

   Sorgeloos, P. 1981.  Availability of reference Artemia cysts. Aquaculture.  Vol. 23, pp. 381-382.

   Vanhaecke, P., P. Lavens, and P. Sorgeloos. 1983. International Study on Artemia, XVII.  Energy consump-
           tion in cysts  and early larval stages of various geographical strains of Artemia. Ann. Soc. R. Zool.
           Belg. Vol. 113, pp. 155-165.

   Vanhaecke, P., and P. Sorgeloos.  1983. International Study on Artemia, XIX.  Hatching data for 10 com-
           mercial sources of brine shrimp cysts and reevaluation of the "hatching efficiency" concept.
           Aquaculture. Vol. 30, pp. 43-52.

   EPA references are available  online at www.epa.gov/npdes.

   If you need additional copies of this document, you can download it at:
           www.epa.gov/npdes/wqbasedpermitting.
                                                                                                  II

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     U.S. ENVIRONMENTAL PROTECTION AGENCY      Sheepshead Minnow (Cypr/nodon voriegotus) and Inland Silverside (Mcnidio bery//ino)
                                                              Larval Survival and Growth Toxicity Test * Supplement to Training Video
                                                    Intentionally Left Blank
12

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    U S' ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon vor/egotus) and Inland Silverside (Menidia beryllina)
                                                   Larval Survival and Growth Toxicity Test  Supplement to Training Video
Glossary
        Acute toxicity. An adverse effect measured in a short period of time (96 hours or less in toxicity tests).
               The effect can be measured in lethality or any variety of effects.

        Algae. Rotifers are fed the algae Tetraselmus suec/ca or Chlorella sp.

        Artemia. The marine invertebrate (referred to as brine shrimp) used as the recommended food source for
               culture and test species; Brazilian or Colombian strains are preferred because the supplies are
               found to have low concentrations of chemical residues and nauplii are of suitably small size.

        Average mean dry weight. All the fish exposed in a given test chamber (replicate) are weighed together.
               Trie total dry weight is divided by the number of surviving fish in the replicate to obtain the
               average mean dry weight.

        Chronic toxicity. An adverse effect that occurs over a long exposure period. The effect can be lethality,
               impaired growth, reduced reproduction, etc.

        Crash. Sudden (overnight) death of cultured organisms in a tank.

        Cyprinodon variegatus. The scientific name for the fish species, sheepshead minnow. The sheepshead
               minnow is a short, deep-bodied, compressed fish. It has large scales and a dark marginal band
               on its tail. It occurs in hypersaline lagoons and connecting channels, and is found on muddy
               bottoms in turbid waters from North and South America: Massachusetts, USA to northeastern
               Mexico; also West Indies; northern coast of South America, Bahamas, Antilles, Gulf of Mexico,
               Yucatan and Venezuela. It is omnivorous, consuming organic detritus and algae, as well as
               microcrustaceans, and  dipteran larvae. Sheepshead minnows are very abundant and easily
               reproduced in captivity.

        Cyst. The life stage of unhatched Artemia.

        Diluent water. Dilution water used to prepare the effluent concentrations.

        Effluent sample. A representative collection of a NPDES permitted facility's discharge that is to be tested.

        Effluent concentration. Different dilutions, or concentrations, of an effluent used to determine the
               biological  effects on test organisms (i.e., inland silversides or sheepshead minnows).

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

        Hypothesis testing. Technique (e.g., Dunnett's test)  that determines what concentration is statistically
               different from the control. Endpoints determined from hypothesis testing are NOEC and LOEC.

        \C25 (Inhibition Concentration, 25%). The point estimate of the toxicant concentration that would cause a
               25% reduction in a non-quantal biological measurement (e.g., reproduction or growth) calculated
               from a continuous model.

        Larvae. Post-hatch fish that are  not free-swimming and are morphologically immature (i.e.,  <24 hr-old).

        LC50 (Lethal Concentration, 50%). The concentration of toxicant or effluent that would cause death to
               50% of the test organisms at a specific time  of observations (e.g., 96-hour LC50).

        Lowest Observed Effect Concentration (LOEC). The LOEC is the lowest concentration of toxicant to
               which organisms are exposed in a test, which causes statistically significant adverse effects on

                                                                                              Glossary-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cvprinodon variegatus) and Inland Silverside (Menidia beryllina)
                                                   Larval Survival and Growth Toxicity Test  Supplement to Training Video
               the test organisms (i.e., where the values for the observed endpoints are statistically significantly
               different from the control). The definitions of NOEC and LOEC assume a strict dose-response
               relationship between toxicant concentration and organism response.

        Minimum Significant Difference (MSD). The MSD is the magnitude of difference from the control where
               the null hypothesis is rejected in a statistical test comparing a treatment with a control. MSD
               is based on the number of replicates, control performance and power of the test. MSD is often
               measured as a percent and referred to as PMSD.

        Menidia beryllina.  The scientific  name for the fish species, inland silverside. It is a marine/estuarine
               species that ascends rivers. In fresh water, inland silverside usually occurs at the surface of clear,
               quiet water over sand or gravel. It feeds on zooplankton and is found in coastal waters from the
               Western Atlantic: Massachusetts to southern Florida in the USA and around the Gulf of Mexico to
               northeastern Mexico.

        Nauplii. Free-swimming microscopic larvae stage characteristic of copepods, ostracods, barnacles, etc.
               typically only with three pairs of appendages.

        No Observed Effect Concentration (NOEC). The NOEC is the highest tested concentration of toxicant to
               which organisms are exposed in a full life-cycle or partial  life-cycle (short-term) test, that causes
               no observable adverse effect on the test organism (i.e., the highest concentration of toxicant
               at which the values for the observed responses are not statistically significantly different from
               the controls). NOECs calculated by hypothesis testing are dependent upon the concentrations
               selected.

        NPDES (National Pollutant Discharge Elimination System) Program. The national program for issuing,
               modifying,  revoking and reissuing, terminating, monitoring, and enforcing permits, and imposing
               and enforcing pretreatment requirements under Sections 307, 38,  402, and 405 of the Clean
               Water Act.

        Point Estimation Techniques. This technique is used to determine the effluent concentration at which
               adverse effects (e.g., fertilization, growth or survival) occurred, such as Probit, Interpolation
               Method, Spearman-Karber. For  example, a concentration at which a 25% reduction  in
               reproduction and survival occurred.

        Receiving Water Concentration (RWC). The RWC is the concentration of a toxicant or the parameter
               toxicity in the receiving water  (i.e., riverine, lake, reservoir, estuary or ocean) after mixing.

        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.

        Rotifer. The rotifer, Brachionus plicatilis is fed to newly-hatched inland silverside larvae until they are  large
               enough to be fed Artemia.

        Static renewal. The daily replacement of effluent medium in the test chamber.

        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.

        Toxicity test. A procedure to measure the toxicity of a chemical or effluent using living organisms. The test
               measures the degree of response of an exposed organism to a specific chemical  or effluent..

        WET (Whole effluent toxicity). The total toxic effect of an effluent measured directly with a toxicity test.
Glossary-2

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 g^ U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia berylllna)
 =/                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
Appendix A:
Preparing  Hypersaline  Brine  (MSB)
       Salinity adjustments are a vital part of using marine and estuarine species for toxicity testing. Because the
       majority of industrial and sewage treatment effluents entering marine and estuarine waters contain little or
       no measurable salts, the salinity of these effluents must be adjusted before exposing estuarine or marine
       plants and animals to the test solutions. It also is important to maintain constant salinity across all treat-
       ments throughout the test for quality control. Finally, matching the test solution's salinity to the expected
       receiving water's salinity may require salinity adjustments. NHEERL-AED uses MSB, prepared from filtered
       natural seawater, to adjust exposure solution salinities.

       MSB has se'veral advantages over artificial sea salts that make it more suitable for use in toxicity testing.
       Concentrated brine derived from natural seawater contains the necessary trace metals, biogenic colloids,
       and some of the microbial components necessary for adequate growth, survival, and/or reproduction of
       test organisms. MSB can  be held for prolonged periods without any apparent degradation, added directly to
       the effluent to increase the salinity, or used as control water by diluting to the desired salinity with deion-
       ized water. The brine can be made from any high-quality, filtered seawater supply through simple heating
       and aerating.

       GENERATING THE BRINE
       The ideal container for making brine from natural seawater has a high surface-to-volume ratio, is made of a
       non-corrosive material, and is easily cleaned. Shallow fiberglass tanks are ideal.

       Thoroughly clean the tank, aeration supply tube, heater, and any other materials that will be in direct
       contact with the brine before adding seawater to the tank. Use a good quality biodegradable detergent, fol-
       lowed by several thorough deionized-water rinses.

       Collect high-quality (and preferably high-salinity) seawater on an incoming tide to minimize the possibility of
       contamination. Special care should be used to prevent any toxic materials from coming in contact with the
       seawater. The water should be filtered to at least 10 urn before placing into the brine tank. Fill the tank with
       seawater, and slowly increase the temperature to 40C. If a heater is immersed directly into the seawater, make
       sure that the heater components will not corrode or leach any substances that could contaminate the brine. A
       thermostatically controlled heat exchanger made from fiberglass is suggested.

       Aeration prevents temperature stratification and increases the rate of evaporation. Use an oil-free air
       compressor to prevent contamination. Evaporate the water for several days,  checking daily (or more or
       less often, depending on  the volume being generated) to ensure that the salinity does not exceed 100%o
       and the temperature does not exceed 40C. If these changes are exceeded, irreversible changes in the
       brine's properties may occur. One such change noted in original studies at NHEERL-AED was a reduction
       in the alkalinity of seawater made from brine with salinity greater than 100%o, and a resulting reduction in
       the animals' general health. Additional seawater may be added.to the brine to produce the volume of brine
       desired.

       When the desired volume and salinity of brine is prepared, filter the brine through a 1-mm filter and pump
       or pour it directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are most
       suitable). Cap the containers, and record the measured salinity and the date generated. Store the brine in
       the dark at room temperature.

       SALINITY ADJUSTMENTS USING HYPERSALINE  BRINE
       To calculate the volume of brine (Vb) to add to a 0%o sample to produce a solution at a desired salinity (Sf),
       use this equation:
                                                                                                   A-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                           Sheepshead Minnow (Cyprinodon vortegatus) and Inland Silverside (Menidio beryllina)
                                                   Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                               vb * sb  = sf * vf
       Where:       Vb =       volume of brine, mL
                    Sb =       salinity of brine, %o

                    Sf =       final salinity, %o
                    Vf =       final volume needed, mL

       Table A-l gives volumes needed to make 20%o test solutions from effluent (0%o), deionized water, and
       100%o HSB. Quantities of effluent, deionized water and a MSB of 100%o (only) needed for conducting daily
       renewals of test solutions at 20%o salinity. The highest concentration achievable is 80% effluent at 20%o
       salinity and 70% effluent at 30%o.                       ,

    Table A-l. Preparation of Test Solutions at a Salinity of 20%> Using HSB for a Final Test Concentration
    Volume of 4000 mL.
Exposure Concentration
80
40
20
10
5
Control
Effluent
(0 %o mL)
3200
1600
800
400
200

Deionized
Water (mL)

1,600
2,400
2,800
3,000
4,000
HSB
(100%.) (mL)
800
800
800
800
800
0
A-2

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 gyl U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegotus) and Inland Silverside (Men/die bery//ino)
 ussX                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
Appendix B:

Preparing Brine  Shrimp and Rotifers for Feeding

       INTRODUCTION
       The brine shrimp (Artemia sp.) is used to feed larval Menidia beryllina and Cyprinodon variegatus in the
       7-day effluent toxicity tests. However, just after hatching, M. beryllina are too small to ingest Artemia, and
       must be fed rotifers (6. plicatilis). Preparation and culture of Artemia and rotifers are described below.

       CULTURING ARTEMIA
       Brine shrimp are highly suited to this testing protocol because: 1) the naupliar stages are nutritionally
       acceptable to these species; 2) they may be obtained from cysts within 24 hours after immersion in seawa-
       ter; and 3) the cysts are readily available and can be stored for prolonged periods of time. There are some
       disadvantages to keep in mind, as well.  For example, it may be difficult to obtain large quantities of cysts.
       In addition, the shrimp's nutritional quality may vary considerably from batch to batch because they are
       obtained from diverse geographical areas.

       Rates offish  growth and survival differed when fed strains of brine shrimp from various geographic loca-
       tions (Klein-MacPhee, et. al., 1982; Johns et al., 1981; Leger and Sorgeloos, 1984).  Therefore, reference
       brine shrimp have been recommended for use in toxicity testing or as a standard for comparison against
       other geographic strains of brine shrimp (Sorgeloos, 1981).

       Brine shrimp normally hatch after incubation for 24 - 48 hours at room temperature. Different geographi-
       cal strains may differ somewhat in time-to-hatch (Vanhaecke and Sorgeloos, 1983) and may diminish in
       nutritional quality after 48 hours (Vanhaecke et al., 1983). Therefore,  it is important to harvest the nauplii
       as soon as possible after approximately 90% have hatched.

       A batch of cysts should be started every 24 hours (for feeding the following day) with the same proportion
       of cysts to seawater so that consistent densities of nauplii are obtained daily (Persoone  et al., 1980).

           1.  Fill a 2- to 4-liter separatory funnel (or other appropriate container) with enough 25 - 30C seawa-
               ter to ensure adequate hatching. Add 10 cc brine shrimp cysts per liter, and aerate for at least 24
               hours at 25C. (Two separatory funnels are recommended, started on alternate days, since it may
               require more than 24 hours to hatch certain strains of brine shrimp.)

           2.  Nauplii will hatch from brine shrimp cysts within 24  -  48 hours,  but before nauplii are fed to the
               fish, they should be separated from the cysts by taking advantage of their phototactic response
               or by straining the culture. After removing the source of air, the nauplii's phototactic response is
               stimulated by covering the top of the funnel with a dark cloth or  paper towel for 5 minutes.  The
               nauplii will concentrate at the bottom. However, leaving nauplii longer than 5 minutes without aer-
               ation may cause mortality. Another way to stimulate phototactic response is to rinse the nauplii
               into a  beaker (500 ml) or a black separator box (15 x 8 x 8 cm high), place a light source at one
               end, and leave for no more than 10 - 15 minutes. After live nauplii migrate toward the light, they
               can be pipetted or siphoned out of the container, leaving the unhatched cysts behind. The nauplii
               can also be separated from the cysts using a sieve.

           3.  Pour the nauplii onto a nylon screen (mesh <150 urn), rinse with filtered control seawater, and
               drain off most of the water.

           4.  On days 0,1, and 2, weigh 4 g (wet weight) or pipette 4 ml of concentrated, rinsed Artemia nau-
               plii from the quantity of Artemia on the screen. On days 3-6, weigh 6  g (wet weight) or pipette
               6 ml nauplii from the quantity of Artemia on the screen. Resuspend the Artemia in 80 ml of sea
               water  in a 100 mL beaker. For days 0-2, the final suspension yields 0.10 g wet weight of Artemia
               nauplii whereas for days 3-6, the final suspension yields 0.15 g wet weight of Artemia nauplii.

                                                                                                   B-l

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    U'S' ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Men/d/o beryllina)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
               Aerate or swirl the Anemia to equally distribute the nauplii; then withdraw and dispense individual
               2 mL portions of Artemia to each test chamber using a pipette or adjustable syringe. Uniform
               distribution of food to all replicates is critical to minimize the variability of larval weight, which is
               important for successful tests. If the replicate chambers are subdivided, divide the 2 ml equally
               among the compartments; if the survival rate of any replicate on any day falls below 50%, reduce
               the volume of Artemia dispensed to that replicate by 2.

       Some live Artemia should remain overnight in test chambers. However, excessive Artemia can decrease DO
       concentrations to below the acceptable limit. Siphon the uneaten Artemia from each chamber prior to test
       solution renewal to ensure that the fish larvae mainly eat newly-hatched nauplii.

       BRINE SHRIMP QUALITY CONTROL
       At a minimum, each batch of purchased brine shrimp should be tested to ensure that they provide the
       nutrients necessary for adequate fish growth. Before use, individual lot numbers of cysts are fed to the
       test organisms in 7-day studies to confirm that the diet is adequate for the purposes of the test. The
       shelf-life of an opened container of cysts may be affected by humidity and temperature, so they should be
       tested each time a test is started. As long as more than 90% of the cysts hatch in 24 - 48 hours and the
       control  responses are acceptable, the cysts may be used (refer to the EPA manual, Short-term Methods for
       Estimating the Chronic Toxicity of Effluents and Receiving Waters in Marine and Estuarine Organisms
       [EPA, 2002a] for acceptability parameters).

       PREPARING  ROTIFER CULTURES (BRACHIONUS PLICATILIS)
       Newly hatched Menidia beryllina larvae are too small to ingest Artemia and must be fed  rotifers
       (Brachionus plicatilis). B. plicatilis can be cultured continuously in  the laboratory when fed algae or yeast in
       10- to 15-L Pyrex carboys at 25C - 28C, 25%o - 35%o salinity. Four 12-L culture carboys, with an outflow
       spout near the bottom, should be maintained simultaneously to optimize production.

       Fill clean carboys with autoclaved seawater. (Alternatively, heat filtered seawater by placing an immersion
       heater in the carboy, and  maintain the temperature to 70C - 80C for 1 hour.)  When the seawater has
       cooled to 25C - 28C, aerate and add a start-up sample of rotifers (50 rotifers/mL) and food (about 1 L
       of a dense algal culture or 0.1 g yeast per liter of seawater). Yeast should be dissolved in a minimum of tap
       water or deionized water before adding it to the culture.

       Check the carboys daily to ensure that adequate food is available  and that the rotifer density is adequate.
       If the water appears clear, add yeast (0.1 g/L) or remove 1 L of water and  replace it with algae. Remove the
       water via the bottom spigot, filtering it through a <60 urn mesh screen. Return any rotifers collected on the
       screen to the culture.

       Keep the carboys away from light to reduce the amount of algae that attaches to the carboy walls. If
       detritus accumulates, populations of ciliates, nematodes, or harpacticoid  copepods that may have been
       inadvertently introduced can rapidly take over the culture. If this occurs, discard the cultures.

       If a precise measure of the rotifer population is needed, resuspend rotifers collected from a known volume
       of water in a smaller volume, preserve them with formalin, and count them in a Sedgwick-Rafter chamber.
       As the density exceeds 50 rotifers/mL, the amount of food per day should be increased to 2 L of algae or
       0.2 g/L of yeast. The optimum density, 300 - 400 rotifers/mL, will be reached  in about 7-10 days and
       should then be cropped daily. This density is sustainable for 2 - 3 weeks. Once that is attained, the rotifers
       should  be cropped daily.

       These rotifers are fed to M. beryllina larvae after hatching until about 5 days old. About 5 days after hatch-
       ing, the larvae can begin feeding on newly hatched Artemia nauplii. They are fed Artemia daily throughout
       the 7-day test.
B-2

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                        Sheepshead Minnow (Cyprinodon variegotus) and Inland Silverside (Menidia beryllina)
                                                Larval Survival and Growth Toxicity Test  Supplement to Training Video
   ALGAL CULTURES
   Algae for feeding the rotifers, Tetraselmus suecica or Chlorella sp., can be cultured in 20-L plastic water
   bottles. Autoclave the bottles (at 110 for 30 minutes) after adding filtered seawater. Cool the bottle to
   room temperature and place them in a temperature controlled chamber at 18C - 20C. Each bottle or
   carboy should contain 1 L of T. suecica or Chlorella sp. starter culture and 100 mL of nutrients.

   The nutrient formula for the algal culture is:
Mix into 12-L deionized water:
Mix on a magnetic stirrer at least 1 hour or until all salts are
dissolved.
Add and stir again:
!80gNaNO3
!2gNaH2PO4
6.l6gEDTA
3.78gFeCI3-6H2O
(Solution should be bright yellow)
Aerate the algal culture vigorously by inserting a pipette through a foam stopper at the top of the bottle or carboy. A
dense algal culture will develop in 7 - 10 days and should be used by day 14. For continuous supply of algal cultures for
rotifer feeding, new cultures should be started every 1 or 2 days. For four 12-L rotifer cultures, 6-8 continuous algal
cultures are needed.
Clean bottles or carboys thoroughly with soap and water, rinsing with deionized water between uses.
                                                                                                    B-3

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     U.S. ENVIRONMENTAL PROTECTION AGENCY      Sheepshead Minnow (C/prinodon variegatus) and Inland Silverside (Menidia beryllina)
                                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                    Intentionally Left Blank
B-4

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    U.S. ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (C/prinodon variegatus) and Inland Silverside (Menidia beryllina)
                                               Larval Survival and Growth Toxicity Test  Supplement to Training Video
Appendix C:

Apparatus  and  Equipment - Sheepshead Minnow and

Inland  Silverside Tests

       Air lines and air stones.  For aerating water containing embryos or larvae, or for supplying air to test
              solutions with low DO.

       Air pump.  For oil-free air supply.

       Balance. Analytical, capable of accurately weighing to 0.00001 g.

       Beakers, six Class A. Borosilicate glass or non-toxic plasticware, 1000 ml for making test solutions,

       Brine shrimp, Artem/a, culture unit.

       Crystallization dishes, beakers, culture dishes (1L), or equivalent. For incubating embryos.

       Desiccator.  For holding dried larvae.

       Dissecting microscope.  For checking embryo viability (Sheepshead test only).

       Droppers, and glass tubing with fire polished edges, 4 mm ID. For transferring larvae.

       Drying oven. 50-105C range, for drying larvae.

       Environmental chamber or equivalent facility with temperature control (25  1 C).

       Facilities for holding and acclimating test organisms.

       Forceps. For transferring dead larvae to weighing boats.

       Inland Silverside culture unit. The test requires approximately 400, 7-11 day old larvae. It is preferable
              to  obtain the test organisms from an in-house culture unit. If it is not feasible to culture fish
              in-house, embryos or larvae can be  obtained from other sources by shipping them in well
              oxygenated saline water in insulated containers.

       Light box.  For counting and observing larvae.

       Meters: pH and DO. For routine physical and chemical measurements.

       NITEX or stainless steel mesh sieves (< 150 um, 500 um, 3-5 mm). For collecting Artem/a naupili
              and fish embryos, and for spawning baskets, respectively.

       Pipet bulbs and filters.  PROPIPET, or equivalent.

       Pipets, automatic. Adjustable, 1 - 100 mL

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

       Pipets, serological. 1-10 ml, graduated.

       Reference weights, Class S.  For checking performance of balance. Weights should bracket the expected
              weights of the weighing paris and the expected weights of the pans plus fish.

       Refractometer. For determining salinity.
                                                                                             C-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                          Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Men/did bery/l/no)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
       Samplers. Automatic sampler, preferably with sample cooling capability, that can collect a 24-hour
               composite sample of 5 L.

       Separatory funnels, 2 L. Two to four for culturing/Artem/a naupili.

       Sheepshead minnow culture unit. The maximum number of larvae required per test will range from a
               maximum of 360, if 15 larvae are used in each of four replicates, to a minimum of 240 per test,
               if 10 larvae are used in each of four replicates.  It is preferable to obtain the test organisms from
               an in-house culture unit.  If it is not feasible to culture fish in-house, embryos or newly hatched
               larvae can be obtained from other sources if shipped in well oxygenated saline water in insulated
               containers.

       Siphon with bulb and clamp.  For cleaning test chambers.

       Standard or micro-Winkler apparatus. For determining DO (optional).

       Test chambers.

               Sheepshead. Four chambers are required for each concentration and the control. Borosilicate
               glass 1000 mL beakers or modified Norberg and Mount (1985) glass chambers used  in the
               short-term inland silverside test may be used. It is recommended that each chamber contain a
               minimum of 50 mL/larvae and allow adequate depth of test solution (5.0 cm).  To avoid potential
               contamination from the air and excessive evaporation of test solutions during the test, the
               chambers should be covered with safety glass plates or sheet plastic (6  mm thick).

               Inland Silverside. Four chambers are required for each concentration and the control. The
               chambers should be borosilicate glass or nontoxic disposable plastic labware. To avoid potential
               contamination from the air and excessive evaporation of test solutions during the test, the
               chambers should be covered with safety glass plates or sheet plastic (6  mm thick).

               Each test chamber for the inland silverside should contain a minimum of 750 mL of test solution.
               A chamber such as the one in Figure C-l constructed of glass and silicone cement has been
               used successfully for this test. This chamber holds an adequate column  of test solution and
               incorporates a sump area where test solutions can be siphoned and renewed without disturbing
               the fragile inland silverside larvae.
               When constructing
               the chamber it
               is recommended
               that the screen be
               a 200-um Nitex
               screen (rather than
               stainless steel) and
               thin pieces of glass
               rods be silicone
               cemented to the
               screen to reinforce
               the bottom and
               sides of the screen
               to create the sump
               area. A minimum
               of silicone should
               be used while sill
               ensuring that the
Figure C-l. Glass test chamber with sump area. Modified from
Norberg and Mount (1985).
                                                 Glass
                                              Reinforcements
                                                              Sump
                                         Source: EPA, 2002a.
C-2

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U S ENVIRONMENTAL PROTECTION AGENCY    Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryl/ina)
                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
           larvae cannot get trapped or drawn into the sump area. All new chambers should be soaked
           overnight in seawater (preferably in flowing seawater) to cure the silicone cement before use.

           Other types of glass chambers can be used such as 1000 ml beakers. However, each chamber
           should contain a minimum of 50 ml of test or control solution per larvae and allow adequate
           depth of test solution (5.0 cm).

   Thermometers. National Bureau of Standards Certified (see EPA 2002a). Used to calibrate laboratory
           thermometers.

   Thermometers, bulb-thermograph or electronic-chart-type. For continuously recording temperature.

   Thermometers, glass or electronic, laboratory grade.  For measuring water temperatures.

   Volumetric flasks and graduated cylinders.  Class A, borosilicate glass or non-toxic plastic labware,
           10 - 1000 ml for making test solutions.

   Wash bottles. For deionized water, for washing embryos from substrates and containers, and for rinsing
           small glassware and instrument electrodes and probes.

   Water purification system. Millipore Milli-Q, deionized water (Dl) or equivalent.
                                                                                               C-3

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     U.S. ENVIRONMENTAL PROTECTION AGENCY      Sheepshead Minnow (Cypr/nodon vor/egatus) and Inland Silverside (Menidia beryl/mo)
                                                              Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                    Intentionally Left Blank
C-4

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    U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cypr/nodon rariegatus) and Inland Silverside (Menldia beryllina)
   ?                                             Larval Survival and Growth Toxicity Test  Supplement to Training Video
Appendix  D:
Reagents and  Consumable Materials
       Buffers, pH 4, pH 7, and pH 10.  (Or as per instructions of instrument manufacturer).  For standards and
              calibration check (see EPA 2002a).

       Data sheets (one set per test). For data recording.

       Ethanol (70%) or formalin (4%). For use as a preservative for the fish larvae.

       Laboratory quality control samples and standards. For calibration of the above methods.

       Markers, waterproof. For marking containers, etc.

       Membranes and filling solutions for DO probe, or reagents. For modified Winkler analysis (see
              EPA 2002a).

       Sample containers.  For sample shipment and storage.

       Tape, colored.  For labeling test chambers.

       Vials, marked. Twenty-four per test, containing 4% formalin or 70% ethanol, to preserve larvae (optional).

       Reference toxicant solutions. Reference toxicants such as sodium chloride (NaCI), potassium chloride
              (KCI), cadmium chloride (CdCI2), copper sulfate (CuS04), sodium dodecyl sulfate (SDS), and
              potassium dichromate (K2Cr207) are suitable for use in the NPDES Program and other Agency
              programs requiring aquatic toxicity tests.

       Reagent water. Defined as distilled or deionized water that does not contain substances which are toxic
              to the test organisms.

       Weighing pans, aluminum. Twenty-four per test (one for each replicate.)
                                                                                               D-l

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     U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Menidia beryl/ma)
                                                               Larval Survival and Growth Toxicity Test  Supplement to Training Video
                                                                                                                      .    ' '   '   '  'Vrf.i.'JPS'io
                                                     Intentionally Left Blank
D-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY   Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Menidio beryllina)
                                           Larval Survival and Growth Toxicity Test  Supplement to Training Video

1?A-!5 ,"-,-, -.-                                         .                 "
Appendix E:

Summary of Test Conditions and Test Acceptability

Criteria

   Table E-l. Summary of Test Conditions and Test Acceptability Criteria for the Sheepshead Minnow,
   Cyprinodon variegatus, Larval Survival and Growth Test with Effluents and Receiving Waters
   (Test Method 1004.0)
Test type
Salinity
Temperature (C)
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number of larvae per test chamber
Number of replicate chambers per
concentration
Number of larvae per concentration
Source of food
Feeding regime
Cleaning
Aeration
Dilution water
Test concentrations
Dilution factor
Test duration
Endpoints
Test acceptability criteria
Sampling requirement
Sample volume required
Static, with 24-hr renewal (required)
20%o - 32%o (maintained at  2%o of the selected test salinity) (recommended)
25C  IC (recommended). Test temperatures must not vary by more than
3C during the test (required)
Ambient laboratory (covered, soft white) light (recommended)
10-20 uE/m2/s (ambient laboratory: 50 - 100 ft-c) (recommended)
16 hr light/8 hr dark (recommended)
600 mL - 1 L containers (recommended)
500 - 750 mL/replicate (loading and DO restrictions must be met)
(recommended)
Daily (required)
Newly hatched larvae (less than 24-hr old; within 24-hr age of each other)
(required)
1 0 (required minimum)
4 (required minimum)
40 (required minimum)
Newly hatched Artemio nauplii (less than 24-hr old) (required)
Feed once per day 0. 10 g wet weight Anemia nauplii per replicate on days
0-2; feed 0.15 g wet weight Artemio nauplii per replicate on days 3-6 (recom-
mended)
Siphon daily, immediately before test solution renewal and feeding (required)
None, unless DO concentration falls below 4.0 mg/L, then aerate all cham-
bers. Rate should be less than 100 bubbles/min. (recommended)
Uncontaminated source of natural seawater, artificial seawater, deionized
water mixed with MSB or artificial sea salts (available options)
Effluent: Five and a control (required). Receiving waters: 100% receiving water
(or minimum of five) and a control (recommended)
Effluents: 50.5 (recommended)
Receiving waters: None, or 50.5 (recommended)
7 days (required)
Survival and growth (weight) (required)
80% or greater survival in controls, average dry weight per surviving organism
in control chambers must be 0.60 mg, if unpreserved or 0.50 mg or greater
average dry weight per surviving control larvae after not more than 7 days in
4% formalin or 70% ethanol (required)
For on-site tests, samples collected daily and used within 24 hr of the time
they are removed from the sampling device.
For off-site tests, a minimum of three samples (e.g., collected on days 1, 3, and
5) with a maximum holding time of 36 hr before first use. (required)
6 L per day (recommended)
   Source: EPA, 2002a, Saltwater Chronic Methods Manual.
                                                                                     E-l

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                             Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia bery//ina)
                                                     Larval Survival and Growth Toxicity Test  Supplement to Training Video
    Table E-2.  Summary of Test Conditions and Test Acceptability Criteria for the Inland
    Silverside, Menidia beryllina, Larval Survival and Growth Test with Effluents and Receiving Waters
    (Test Method 1006.0)
Test type
Salinity 
Temperature (C)
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number of larvae per test chamber
Number of replicate chambers per
concentration
Number of larvae per concentration
Source of food
Feeding regime
Cleaning
Aeration
Dilution water
Test concentrations
Dilution factor
Test duration
Endpoints
Test acceptability criteria
Sampling requirement
^
Sample volume required
Static, with 24-hr renewal (required)
5%o - 32%o (maintained at  2%o of the selected test salinity) (recommended)
25C  IC (recommended). Test temperatures must not vary by more than
3C during the test (required)
Ambient laboratory (covered, soft white) light (recommended)
10-20 pE/m2/s (ambient laboratory: 50 - 100 ft-c) (recommended)
16 hr light/8 hr dark (recommended)
600 mL - 1 L containers (recommended)
500 - 750 mL/replicate (loading and DO restrictions must be met)
(recommended)
Daily (required)
7-11 days post-hatch; within 24-hr age of each other (required)
1 0 (required minimum)
4 (required minimum)
40 (required minimum)
Newly hatched Artemio nauplii; survival of 7-9 day old M. beryllina larvae
improved by feeding 24-hr old Anemia (required)
Feed 0.10 g wet weight Artemia nauplii per replicate on days 02; feed 0.15 g
wet weight Artemia nauplii per replicate on days 3-6 (recommended)
Siphon daily, immediately before test solution renewal and feeding (required)
None, unless DO concentration falls below 4.0 mg/L, then aerate all cham-
bers. Rate should be less than 100 bubbles/min. (recommended)
Uncontaminated source of natural seawater, artificial seawater, deionized
water mixed with MSB or artificial sea salts (available options)
Effluent: Five and a control (required). Receiving waters: 100% receiving water
(or minimum of five) and a control (recommended)
Effluents:  0.5 (recommended).
Receiving waters: None, or ^ 0.5 (recommended)
7 days (required)
Survival and growth (weight) (required)
80% or greater survival in controls, 0.50 mg average dry weight of control
larvae where test starts with 7-day old larvae and dried immediately after
test termination, o_r 0.43 mg or greater average dry weight per surviving
control larvae, preserved not more than 7 days in 4% formalin or 70% ethanol
(required)
For on-site tests, samples collected daily and used within 24 hr of the time
they are removed from the sampling device. For off-site tests, a minimum of
three samples (e.g., collected on days 1, 3, and 5) with a maximum holding
time of 36 hr before first use. (required)
6 L per day (recommended)
    Source: EPA, 2002a. Saltwater Chronic Methods Manual.
E-2

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                         Sheepshead Minnow (Cyprinodon variegotus) and Inland Silverside (Menidia beryllina)

                                                 Larval Survival and Growth Toxicity Test  Supplement to Training Video
Appendix F:  Data  Sheets

    Figure F-l. Data Form for the Sheepshead Minnow and Inland Silverside, Larval Survival and Growth

    Toxicity Test.  Daily Record of Larval Survival and Test Conditions.
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-------
fjirt U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina)

^u^                                                    Larval Survival and Growth Toxicity Test  Supplement to Training Video
    Figure F-l (continued). Data Form for the Sheepshead Minnow and Inland Silverside, Larval Survival and

    Growth Toxicity Test. Daily Record of Larval Survival and Test Conditions..
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F-2

-------
 U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon vor/egotus) and Inland Silverside (Men/dm beryl/ino)
                                                  Larval Survival and Growth Toxicity Test  Supplement to Training Video
Figure F-2. Data Form for the Sheepshead Minnow and Inland Silverside, Larval Survival and Growth
Toxicity Test. Summary of Test Results
    Test Dates:
. Species:
    Effluent Tested:
Treatment
No. Live
Larvae
Survival
(%)
Mean Dry
Wt/Larvae
(mg)  SD
Signif. Diff.
from Control
(o)
Mean Temp.
(C)  SD
Mean Salinity
%0SD
Ave. DO
(mg/L)  SD
















































   Comments:
Source: EPA, 1987a.
                                                                                                       F-3

-------
    U.S. ENVIRONMENTAL PROTECTION AGENCY     Sheepshead Minnow (Cyprinodon voriegotus) and Inland Silverside (Men/din beryl/ino)
                                                     Larval Survival and Growth Toxicity Test  Supplement to Training Video
    Figure F-3.  Data Form for the Sheepshead Minnow and Inland Silverside, Larval Survival and Growth
    Toxicity Test.  Dry Weights of Larvae.
       Test Dates:
                          Species:.
         Pan.
         No.
Cone. &
  Rep.
Initial Wt.
Final Wt.
  (mg)
Diff.
  No.
Larvae
 Avg. Wt./
Larvae (mg)
    Source: EPA, 1987b.
F-4

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If you need additional copies of this document, you can download it at:
             www.epa.gov/npdes/wqbasedpermitting

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               If you need additional copies of this document,
you can download it at: http://cfpub.epa.gov/npdes/wqbasedpermitting/wet.cfm

-------