Whole Effluent Toxicity Training
Video Series
Freshwater Series

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WHOLE EFFLUENT TOXICITY • TRAINING VIDEO SERIES
         Cehodaphnia Survival and
         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-G-06-001
December 2006

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                         NOTICE

The revision of this report has been funded wholly or in part by the
  Environmental Protection Agency under Contract EP-C-05-046.
Mention of trade names or commercial products does not constitute
           endorsement or recommendation for use.

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U.S. ENVIRONMENTAL PROTECTION AGENCY                     Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                        Supplement to Training Video
  This report serves as a supplement to the video "Ceriodaphnia Survival and Reproduction
  Toxicity Tests" (EPA, 2006a). The methods illustrated in the video and described in this report
  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 Freshwater
  Organisms, Fourth Edition (2002a) referred to as the Chronic Methods Manual. The video and
  this report provide details on initiating, renewing, and terminating tests based on the expertise
  of the personnel at the EPA's Mid-Continent Ecology Division (MED) in Duluth, Minnesota (EPA-
  Duluth).

  This report and its accompanying video are part  of a series of training videos produced by EPA's
  Office of Wastewater Management. The videos entitled "Fathead Minnow (Pimephales promelas)
  Larval Survival and Growth Toxicity Tests" and  "Culturing of Fathead Minnows (Pimephales
  promelas)" (EPA, 2006b,c) complement the material in this video by explaining the method
  for testing and culturing fathead minnows for use in freshwater whole effluent toxicity tests.
  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                           Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                                             Supplement to Training Video
                                             Intentionally Left Blank

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                                                 rfdpliiiio Survival and Reproduction Toxieity Tests
                                                                 Supplement to Training Video
Foreword	i

Introduction	1

Background	1

Test Method	1

Culturing	1

Effluent Sampling	2

Dilution Preparation	2

Test Organisms	2

Routine Chemistries	3

Feeding	3

Renewal	4

Test Termination	4

Test Acceptability and Data Review	5

Other Procedural Considerations	5

Food Preparation	5

Culturing Techniques	6

Diluent Water	7

Additional Requirements	7

References	8

Glossary	Glossary-1

Appendix A: Summary of Test Conditions and Test Acceptability Criteria	A-l

Appendix B: Reagents & Consumable Materials	B-l

Appendix C: Apparatus & Equipment List	C-l

Appendix D: Food Preparation	D-l

Appendix E: Ceriodaphnia dubia Anatomy	E-l

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 NVIRONMENTAL PROTECTION AGENCY                    Ceriodaphnia      and
                                                                Supplement to Training Video
TABLE

 Table 1. Monitoring Schedule.
FIGURE

 Figure 1. Test board as randomized for test concentrations and adding young
         to begin test	3

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 O • U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                        Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                            Supplement to Training Video
Introduction

      This report accompanies the Environmental Protection Agency's video training for conducting
      Ceriodaphnia dubia (freshwater water flea) survival and reproduction toxicity tests (EPA, 2006a).
      The test method is found in Short-term Methods for Estimating the Chronic Toxicity of Effluents
      and Receiving Waters to Freshwater Organisms, Fourth Edition (EPA, 2002a). The test is adapted
      from methods developed by Dr. Donald Mount and Teresa Norberg-King of EPA's Mid-Continent
      Ecology Division (MED), in Duluth, Minnesota. The material presented in both the video and this
      report 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 Glean 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 permit limitations control pollutant discharges on a whole effluent toxicity (WET)
      basis, rather than by a chemical specific approach.
  The test method requires a static renewal
  exposure. Every 24 hours, the Ceriodaphnia
  are transferred to  a new test chamber
  containing a freshly prepared solution of the
  appropriate effluent concentration.
                                         The test method requires a static renewal exposure sys-
                                         tem (Appendix A). Every 24 hours the Ceriodaphnia dubia
                                         are transferred to a new test chamber containing a fresh-
                                         ly prepared solution of the appropriate effluent concentra-
                                         tion. This report covers the general procedures used for
                                         conducting Ceriodaphnia toxicity tests. The Ceriodaphnia
                                         short-term chronic test estimates the toxicity of an efflu-
      ent by exposing test organisms to different concentrations of effluent. The test results are based
      on survival and reproduction of the organisms using static renewal system. This report and the
      accompanying video describe how the test is set up, initiated, monitored, renewed, and termi-
      nated, followed by suggestions on maintaining healthy test organisms.

Test  Method

      GULTURING
      The first step is to set up Ceriodaphnia cultures that produce the young, called neonates, for
      the test. Mass culturing can be used to maintain large laboratory stocks, but individual brood
      cultures must be started at least three weeks before brood animals are needed for testing. The
                                   survival records of these individual brood cultures must be
                                   maintained to assure that healthy animals are used for testing.
                                   Brood animals are fed daily and the culture media is renewed at
                                   least three times a week.
  Cultures produce Brood animals that
  produce Test animals.
      For a test with 5 concentrations of effluent and a control, with 10
      replicates per concentration, 10 brood cups with 8 or more young
      of the right age are randomly selected from a brood board. Test
      organisms must be less than 24 hours old and within 8 hours of
      the same age. Using neonates produced within 2 or 4 hours of each
      other is even more desirable. In order to obtain  a sufficient num-
      ber of test animals within this age specification, it is advisable to
      have at least 50 to 60 brood animals to select from. Neonates must
      be taken only from adults in individual cultures that have eight or
                                                                      Five effluent concentrations
                                                                      + 1 control
                                                                      = 6 concentrations
                                                                      X  10 replicates
                                                                      = 60 test vessels
                                                                      X  I brood animal/replicate
                                                                      = 60 brood animals

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C • U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                      Ceriodaphnia Sur¥ival and Reproduction Toxicity Tests
                                                                          Supplement to Training Video
     more young in their third or subsequent brood. Adults in these individual brood cultures can be
     used as brood stock until they are 14 days old.

     EFFLUENT SAMPLING
     Effluent sampling should be conducted according to the Chronic Methods Manual (EPA, 2002a)
     and any conditions specified in a regulatory permit. Samples are collected over a 24 hour period
     or when a 24 hour composite sampling period is completed. If the data from the samples are to
                                        be acceptable for use in the NPDES program, the lapsed
                                        time (holding time) from sample collection to first use of
                                        each grab or composite sample must not exceed 36 hours.
                                        However, in no case should more than 72 hours lapse
                                        between collection and first use of sample. In the USEPA
                                        Federal register notice (2002b), EPA clarified the allowable
                                        sample holding temperatures for WET samples as 0 - 6°G
                                        and clarified that hand-delivered samples used on the day
                                        of collection do not need to be cooled to 0 - 6°G prior to
                                        test initiation.
Samples must be collected as described in
the methods manual or as required by a
permitting authority. Samples cannot be
older than 72 hours for first use. Samples
may be used for renewal at 24, 48, and/or
72 hours after first use.
     DILUTION PREPARATION
     Warm the effluent to 25 ± 1°C slowly to avoid exceeding the desired temperature. Maintain
     this temperature throughout the test. Once the effluent and the dilution water have reached the
     desired temperature, the dilutions can be prepared. EPA recommends the use of five exposure
     concentrations and a control for each test using a 0.5 dilution series, with  10 replicates per con-
     centration, 15 ml of medium and one organism per replicate. Sufficient test solution, for example
     500 mL, should be prepared at each concentration to provide additional volume for chemical
     analyses of each test concentration and the control.

     TEST ORGANISMS
     On the day of the test, neonates for testing are obtained by removing any young from the culture
     beakers or transferring the brood animals to new beakers early in the morning on the day the
     test begins. Separate the brood cups with at least 8 young per female by 2- to 4-hours blocks,
     until at least 10 brood cups have sufficient young. If the neonates are held more than 1- to 2-
     hours before being used in the test, they should be fed appropriate amounts of the yeast, cereal
     leaves and trout food (YCT) and algal concentrate. (See feeding section below.) Be sure to record
     the age range of test organisms, the source of the neonates, and any feeding that occurs on test
     data sheets. If a brood animal is not producing three broods in seven days with 8 to  14 young in
     the brood, it may be overcrowded, underfed, or stressed in some way.

     The recommended method for conducting the test is to use a randomized block design of treat-
     ment. This is done by randomly selecting 10 brood females with more than 8 young each. The
     neonates from each female are then assigned to one replicate of each effluent concentration.
     This way,  each replicate of all of the concentrations is conducted with a neonate from a different
     brood animal. This procedure allows for tracking the performance of the young from each female
     (Figure 1). Also, if a female produces one weak offspring,  or male, the likelihood of producing all
     weak or all male offspring is greater. Therefore, by using this known parentage technique, poor
     animal performance can be  omitted from all concentrations for one female's young, thus decreas-
     ing variability among replicates. When using the randomized block designed, test chambers are
     randomized once at the beginning of the test.

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C  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                        Ceriodaphnia Sur¥i¥ai and Reproduction Toxicity Tests
                                                                            Supplement to Training Video
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Sample
Concentration Key:
1 = Control
1 = 6.25% effluent
3= 12.5%
5 = 50%
6 = 100%
             Figure I. Test board as randomized for test conecentrations and adding young to begin test.

     ROUTINE CHEMISTRIES
     Once the test concentrations are prepared, set aside an aliquot of each for the routine chemistries
     that must be performed. This allows for the chemistries to be performed without contaminating
     the actual test solutions with the probes. For test initiation and renewals, measure and record
                                  the dissolved oxygen (DO) at the beginning of each 24 hour renew-
                                  al in each test concentration. This ensures that the dilutions are
                                  not supersaturated or below 4.0 mg/L. If they are, they should be
                                  aerated gently for a short period of time. DO measurements also
                                  should be performed at the end of each 24 hour exposure period
                                  for one replicate in each concentration and the control.
EPA recommends that the
test temperatures be recorded
continuously during the test.
Temperature should be monitored
continuously or observed and
recorded daily for at least two
locations in the environmental
control system or the samples.
                                  DO, temperature, pH, and total residual chlorine must be mea-
                                  sured on each new sample. EPA also recommends that total alka-
                                  linity, total hardness, and conductivity be measured on each new
                                  sample (Appendix C). DO, temperature, andpH are measured at
                                  the beginning and end of each 24 hour renewal in at least one test
                                  chamber for each test concentration and in the  control. Measuring
     conductivity at the beginning and end of each 24 hour renewal is preferred but not required. The
     temperature and pH of the effluent sample also must be measured each day before preparing the
     test solutions (Table 1).
     FEEDING
                                                                See Other Procedural
                                                                Considerations and Appendix D
                                                                for Food Preparation Instructions
     The test animals are fed daily with the same food that the
     cultures receive. At test initiation and at the time of the
     daily effluent renewal, 0.1 mL of the YCT food mixture and
     0.1 mL of algae are added to each 15  mL of test medium.
     The YCT food mixture is made of yeast, cereal leaves, and
     trout food (Appendix D).  The algae is Pseudokirchneriella subcapitata (former species name was
     Selenastrum capricornutum). In the 15 mL test volume, the number of algae cells for feeding
     should be approximately 2 to 2.3 x 105 cells per mL.

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e  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                        Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                             Supplement to Training Video
      Table I. Monitoring Schedule
Parameter
Dissolved oxygen1-2
Temperature1-2-3
pH1-2-3
Conductivity1-2
Alkalinity1
Hardness1
Total Residual Chlorine1
Ammonia4
/Monitoring Frequency
Each New Sample
X
X
X
X
X
X
X
X
24 hour Exposure Period
Beginning
X
X
X
X
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 Measured in the effluent sample each day before preparation of new test solutions.
      4 Iftoxicity may be contributed by total ammonia (> Smgll), ammonia is measured on 100% effluent.

      Between renewals, cover the test cups to prevent evaporation, as well as possible contamination
      from the laboratory environment. Glass is recommended because plexiglass can warp with
      moisture. Place the test trays in a temperature and photoperiod-controlled room or chamber
      and conduct the test at a temperature of 25° ± 1°C. Maintaining the temperature is important
      due to possible effects on reproduction. Control the photoperiod of 16 hours light and 8 hours
      dark. Also, light intensity and quality should be at a minimum of standard ambient laboratory
      conditions.

      RENEWAL
      Once the test is initiated, the renewals on days two through six are performed in the same man-
      ner as the test start. To do the daily renewals, prepare the test solutions, distribute the solutions
      to clean test containers using the same randomization layout used at test initiation and add food
      to each container. Test animals are then transferred with a glass dropper or pipette, releasing
      the animal under the surface of the water. As young may be present on day three, exercise care
      to transfer only the adult and not the young and  do not dispose of the test cups until the young
      have been counted. If a test animal is injured or killed during the renewal process, it should
      be  discarded and recorded. By killing one animal, you have reduced the initial number of test
      organisms for that treatment by one.

      Record observations daily on Ceriodaphnia survival and reproduction. Three or four broods
      are usually obtained from the control animals during the six to eight-day test period, provided
      that the control water is  reliable water. The first  brood of 3 to 5 young is usually released on
      day three or four, with successive broods released every 30 to 36 hours thereafter. When count-
      ing the young, organisms are observed more readily if viewed against a black background or by
      using a low power dissecting microscope. When using a microscope, a 15 ml water volume in the
      beaker or plastic cup allows viewing the entire water column without adjustments to the micro-
      scope. Using a bottom light source also helps.

      TEST TERMINATION
      In the absence of toxicants, adults typically will  produce an average of 15 to 35 young in the
      first three broods. Some effluents or toxicants may cause the young to be aborted. Tests should
      be terminated when 60% or more of the surviving control females have produced their third
      brood, or at the end of 8 days, whichever occurs first. Because of the rapid rate of development of
      Ceriodaphnia, complete all observations on organism survival and numbers of offspring within
      2 hours of test termination.

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 O • U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                       Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                           Supplement to Training Video
      For the test to be considered acceptable, the control animals must have a survival rate of at
      least 80% or better, produce an average of 15 young per surviving female, and 60% of surviving
      control females must have produced three broods. In this three-brood test, offspring from fourth
      or higher broods should not be counted or included in test results. Statistical analyses of the test
      results should be conducted according to the Chronic Methods Manual (EPA, 2002a).

      TEST ACCEPTABILITY AND DATA REVIEW
      Test data are  reviewed to verify that test acceptability criteria (TAG) requirements for a valid
      test have been met. For instance, the TAG requires 80% or greater survival in controls with 60%
                                         of surviving  organisms producing 3 broods of at least 15
                                         total neonates. Any test not meeting the minimum TAG is
                                         considered invalid. All invalid tests must be repeated with
                                         a newly-collected sample. Further guidance is provided in
                                         the Chronic Methods Manual (EPA, 2002a).
Data analysis procedures are presented
in the appendices of the Chronic Methods
Manual (EPA, 2002a)
      The test results must be reviewed for concentration-response relationships for all multi-concen-
      tration tests. The concentration-response relationship generated for each multi-concentration test
      must be reviewed to ensure that calculated test results are interpreted appropriately. In conjunc-
      tion with this requirement, EPA has provided recommended guidance for concentration-response
      relationship review (EPA, 2000a).

      EPA's promulgated toxicity testing method manual recommends the use of point estimation tech-
      nique 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., LG50, IC25) analyses are not used. EPA specifies the PMSD must be calculated
      when NPDES permits require sublethal hypothesis testing. To reduce the within-test variability
      and to increase statistical sensitivity when test endpoints are expressed using hypothesis testing
      rather than the preferred point estimation techniques, EPA also requires that variability crite-
      ria be applied as a test review step when NPDES permits require sublethal hypothesis testing
      endpoints (i.e., no observed effect concentration [NOEG] or lowest observed effect concentration
      [LOEG]) and the effluent has been determined to have no toxicity at the permitted receiving water
      concentration (EPA, 2002a).

Other  Procedural Considerations

      FOOD PREPARATION
      In addition to strict adherence to the test protocol, there are other factors that may influence test
      results. The first is food preparation (see Appendix E - Food Preparation). It is important that the
      YGT  mixture is similar in composition from week to week. To prepare the food, follow the gen-
      eral guideline in the Chronic  Methods Manual,  which is briefly described here:

      Trout Food is prepared first. Five grams of trout food starter granules or Vs" pellets are added to
      1 liter of MILLI-Q® or equivalent water and mixed well in a blender. Digest prior to use by aerat-
      ing continuously from the bottom of the vessel for one week at ambient laboratory temperature.
      Once the trout food has been  digested, place in a refrigerator and allow it to settle for a mini-
      mum of 1 hour. The supernatant is then filtered through a Nytex® 110 mesh to remove the larger
      particulates. Aliquots of this  food are used fresh or stored in the freezer for later use.

      Cereal leaves of the YCT are prepared 24 hours in advance. Put 5.0 g of cereal leaves in 1 liter
      of the same diluent water, e.g., MILLI-Q®, as was used for the trout chow fermentation. Mix in
      a blender at high speed for 5 minutes, or stir on a stir plate for 24 hours at a moderate rate. If a

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_£ U.S. ENVIRONMENTAL PROTECTION AGENCY                     Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                           Supplement to Training Video
      blender is used to suspend the material, place in a refrigerator overnight to settle. If a stir plate
      is used, allow to settle for 1 hour. Decant and filter the cereal leaves through a Nytex® 110 mesh.
      Cereal leaves are a powder of dehydrated cereal or grass leaves with Vitamin A, B2, C, and K.

      Yeast for the YCT is mixed in the same diluent water as the other food items. Five grams of yeast
      should be placed in 1 liter of the MILLI-Q® water and mixed with a blender at low speed, shaked
      vigorously by hand, or stirred with a magnetic stirrer until the yeast is well  dispersed. The yeast
      should be made up on the same day that all of the components are put together and added while
      in suspension.

      After all three components are ready, mix equal volumes together and shake well. Measuring
      and adjusting suspended solids in the YCT (1.7 - 1.9 g solids/L) ensure that the food level is
      similar from batch to batch. Freshly prepared food can be used immediately  (a maximum of two
      weeks if stored in the refrigerator between feedings), or it can be frozen until needed (not more
      than 3 months).

      Algae is fed as a supplement to the YCT. For culturing the Pseudokirchneriella subcapitata (for-
      merly Selenastrum capricornutum), the media from the Chronic Methods Manual is recommend-
      ed. Again, the cell density of the algae must be monitored with each batch by using an electronic
      particle counter, a microscope and hemacytometer, fiuormeter, or spectrophotometer. The algae
      cells should be monitored occasionally to be sure that the culture is not contaminated with any
      other types of algae. Algal concentrate may be stored in the refrigerator for two weeks.

      GULTURING TECHNIQUES
      Culturing the test organisms is an important factor for accurate  testing. To provide cultures
      of overlapping ages, new cultures are started weekly, using neonates from adults who produce
      at least eight young in their third or fourth brood. The adults can be used as  sources of neo-
      nates until 14 days of age. A minimum of two ages of cultures are maintained concurrently to
      provide backup supplies of organisms in case of problems. Cultures which are properly main-
      tained should produce at least 20 young per adult in three broods (seven days or less). Typically,
      60 adult females will produce more than the minimum number of neonates (120) required for
      two tests. Records should be maintained on the survival of brood organisms  and number of off-
      spring at each renewal. Greater than 20% mortality of adults or less than  an  average of 20 young
      per female would indicate problems, such as poor quality of culture media or food. Cultures that
      do not meet these criteria should not be used as a source of test organisms.

      Low production  during the three-brood test may be due to the health of the cultures, or fac-
      tors such as the techniques of the lab personnel. One factor may  be unfamiliarity of laboratory
      personnel with culturing techniques. Another may be that the brood animals were not adequate
      for producing test animals. To avoid these problems, a great deal of effort should be put into
      culturing. EPA recommends that laboratories maintain records on the brood animals to monitor
      their health. Count young production for seven days each month  for 10 animals in the culture to
      determine how well the culture is performing.

      It is also important that cultures are fed daily and that cultures  are routinely changed (e.g., on
      Monday, Wednesday, and Friday) or the animals will quickly become overcrowded with their
      own young. A general guideline is to put as much effort into culturing as into testing because the
      success of the test relies on good culturing techniques.

      Keep in mind that mass cultures are used only as a "backup" reservoir of organisms. If these ani-
      mals are used as the source of brood organisms for individual culture, they should be maintained
      in good condition by frequent renewal with new culture medium at least twice a week for two
      weeks.

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C  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                       Ceriodaphnia Sur¥i¥al and Reproduction Toxicity Tests
                                                                           Supplement to Training Video
     EPA recommends that chronic toxicity tests be performed monthly with a reference toxicant.
     Ceriodaphnia neonates, less than 24 hours old and all within 8 hours of the same age, are used
     to monitor the chronic toxicity of the reference toxicant to the Ceriodaphnia produced by the
     culture unit (see the Chronic Methods Manual for specific requirements).
                                                                 Testing laboratory must perform
                                                                 at least one acceptable reference
                                                                 toxicant test per month for each
                                                                 type of toxicity test method
                                                                 conducted in that month.
Records that should be maintained are:
    •   source of organisms used to start the cultures

    •   type of food and feeding times

    •   dates culture were thinned and restarted

    •   rate of reproduction in individual cultures

    •   daily observations of the condition and behavior of the organisms in the cultures, and

    •   dates and results of reference toxicant tests performed.

DILUENT WATER
Another factor that is important to the success of the test is the choice of water to use as the
diluent. Not all surface water is reliable for culturing. It is  important to establish what the
young production and survival rates are for each water that is used, before initiating a test.
Maintaining survival records on the brood  animals is helpful. To determine how well a cul-
ture performs, count young production for seven days each month for 10 animals in the  culture.
For artificially reconstituted waters, it is very important to start with a "high purity" distilled,
deionzed or carbon filtered water. To achieve this install a high grade filtering system and install
the filters in the following order:
                             •   ion exchange

                             •   ion exchange

                             •   carbon filter

                             •   Organex-Q®

Follow these with a final bacteria filter. Also, do not store water for more than 14 days.

Finally, if an adult does not produce young  during the test, it should be put on a slide and its
sex determined. This is relatively easy and is to be performed according to the guidelines in the
Chronic Methods Manual.

ADDITIONAL REQUIREMENTS
Test review is an important part of an overall quality assurance program and is necessary for
ensuring that all test results are reported accurately. Test review should be conducted on each
test by both the testing laboratory and the regulatory authority. To do this, the collection and
handling of samples are reviewed to verify that the sampling and handling procedures were
followed. For WET test data submitted under NPDES permits, all required test conditions must
be met or the test is considered invalid and must be repeated with  a newly collected sample.
The Chronic Methods Manual (EPA, 2002a)  provides some guidance on how to handle deviations
from recommended test conditions that must be evaluated  on a case-by-case basis to determine
the validity of test results. Chain-of-custody forms are reviewed to verify that samples were
tested within allowable sample holding times. Any deviations from the procedures are to be
documented and described in the data report. Next, the test data are reviewed to verify that

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US, ENVIRONMENTAL PROTECTION AGENCY                     Ceriodophnia Survival and Reproduction Toxicity Tests
                                                                        Supplement to I raining Video
  TAG requirements for a valid test have been met. Any test not meeting the minimum test
  acceptability criteria is considered invalid. All invalid tests must be repeated with a newly
  collected sample. The test conditions are reviewed and compared to the specifications listed
  in the summary of test condition tables (Appendix A). Physical and chemical measurements
  taken during the test (e.g., temperature, pH, and DO) are reviewed and compared to specified
  ranges. Any deviations from TAG specifications should be documented and described in the
  data report. The statistical methods used for analyzing test data are reviewed to verify that
  the recommended flowcharts for statistical analysis were followed. Any deviation from the
  recommended flowcharts for selection of statistical methods should be noted in the data report.

  EPA recommends control charts be prepared for each combination of reference toxicant, test
  species, test conditions, endpoints, and for the PMSDs calculated for successive effluent tests
  (EPA, 2000). The TAG, test conditions, concentration-response relationship, and test sensitivity of
  the reference toxicant test are reviewed to verify that the reference toxicant test conducted was
  valid. Toxicity endpoints from five or six tests are adequate for establishing the control charts.
  Laboratories should compare the calculated coefficient of variation, GV (i.e., standard deviation/
  mean) of the IG25 for the 20 most recent  data points to the distribution of laboratory GVs reported
  nationally for reference toxicant testing.
   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, DC. EPA 833-R-00-003.

   EPA, 2000b. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing
          (40GFRPart 136 [FRL-7069-7]). Office of Water, Cincinnati, OH. EPA/821-R-02-013.

   EPA, 2002a. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
          Waters to Freshwater Organisms. Fourth Edition. Office of Water, Cincinnati, OH.
          EPA/821-R-02-013.

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

   EPA, 2006a. Ceriodaphnia Survival and Reproduction Toxicity Test. Video Tape and
          Supplemental Report. EPA-833-C-06-001. December 2006.

   EPA, 2006b. Fathead Minnow Larval Survival and Growth Toxicity Test, Training Video.
          EPA-833-C-06-001. December 2006.

   EPA, 2006c. Culturing Fathead Minnows (Pimephales promelas), Training Video.
          EPA-833-C-06-001. December 2006.

   References are available online at www.epa.gov/npdes.

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 j5  U.S. ENVIRONMENTAL PROTECTION AGENCY                      Ceriodaphnia Survival and Reproduction 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.

       Algae.  The algae used is Pseudokirchneriella subcapitata (former species name Selenastrum
              capricornutum).

       Cereal Leaves. A powder of dehydrated cereal or grass leaves used in the food mixture.

       Ceriodaphnia. The  freshwater invertebrate cladoceran, Ceriodaphnia dubia (water flea), used for
              this test method.

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

       Coefficient of Variation (CV) is a standard statistical measure of the relative variation of
              a distribution or set of data, denned as the standard deviation divided by the mean.
              Coefficient  of variation is a measure of precision within (intralaboratory) and among
              (interlaboratory) laboratories.

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

       Effluent concentrations. Concentrations or dilutions of an effluent sample to which
              Ceriodaphnia 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
              areNOEC andLOEC.

       Inhibition Concentration (1C). 1C is a point estimate of the toxicant concentration that would
              cause a given percent reduction (i.e., IC25) in a non-quantal biological measurement (e.g.,
              reproduction or growth) calculated from a continuous model.

       Lethal  concentration (i.e.  LC50). The toxicant concentration resulting in a reduction of survival
              equal to 50% of the exposed organisms at a specific time of observation.

       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 MSB 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.
                                                                                      Glossary-1

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 jC  U.S. ENVIRONMENTAL PROTECTION AGENCY                      Ceriodaptmia Sur¥i¥al and Reproduction Toxieity Tests
                                                                              Supplement to Training Video
       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.

       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.

       Neonate. Newly released Ceriodaphnia (less than 24 hours old) used for testing.

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

       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.

       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.

       Trout food. The trout food can be flakes or pellets, and should follow the TJSFWS Specifications.

       Whole effluent toxicity (WET). The aggregate toxic effect of an effluent measured directly
              with a toxicity test.

       Yeast. The yeast used for feeding is common dry yeast.
GIossary-2

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                               Ceriodaphnia Survival and Reproduction Toxieity Tests
                                                                Supplement to Training Video
Appendix A
     SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
Test type
Temperature (°C)
Light quality
Light intensity
Photoperiod
Test vessel size
Test solution volume
Renewal of test concentrations
Age of test organisms
No. neonates per test chamber
No. replicate test chambers
per concentration
No. neonates per test
concentration
Feeding regime
Cleaning
Aeration
Dilution water
Test Solutions
Dilution factor
Test duration
Effects measured
Test acceptability
Sampling requirements
Minimum sample volume daily
Static renewal (required)
25° ± I°C recommended test temperature should not deviate (i.e.,
maximum minus minimum temperature) by more than 3°C during the
test (required)
Ambient laboratory illumination (recommended)
10-20 uE/m2/s, or 50-100 ft-c (ambient laboratory levels)
(recommended)
16 hours light, 8 hours darkness (recommended)
30 mL (recommended minimum)
15 mL (recommended minimum)
Daily (required)
Less than 24 hours; and all released within a 8-hour period (required)
\ assigned using blockings by known parentage (required)
\ 0 (required minimum)
1 0 (required minimum)
Feed O.I ml each of the YCT and algal suspension per test chamber
daily (recommended)
Use freshly cleaned glass beaker or new plastic cups daily
(recommended)
None (recommended)
Uncontaminated source of receiving or other natural water, synthetic
water prepared using Millipore Milli-Q® or equivalent deionized water
and reagent grade chemicals or dilute mineral water (available options)
Effluents: 5 and a control (required minimum)
Receiving water: 100% receiving water (or a minimum of 5) and a
control (recommended)
Effluents: >0.5 (recommended)
Receiving waters: None or > 0.5 (recommended)
Until 60% or more of surviving control females have three broods
(maximum test duration 8 days) (required)
Survival and reproduction (required)
80% or greater survival of control organisms and an average of > 15
or more young per surviving female in the control solutions. 60% of
surviving control females must produced three broods (required)
For on-site tests, samples collected daily and used within 24 hours
of the time they are removed from the sampling device. For off-site
tests, a minimum of three 24 hour composite samples with a maximum
holding time of 36 hours before first use. (required)
\ L/day (recommended)
                                                                            A-1

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 _£_ - U.S. ENVIRONMENTAL PROTECTION AGENCY                    Ceriodaphnia Sur¥i¥al and Reproduction Toxicity Tests
                                                                           Supplement to Training Video
                 B

      REAGENTS AND CONSUMABLE MATERIALS

       Algae - Starter cultures of the green algae are available from commercial suppliers.

       Data Sheets (one set per test) - for recording the data.

       Effluent, surface water, and dilution water - Dilution water that contains undesirable organ-
              isms, that may attack the test organisms should be filtered through a fine mesh net (60-
              um or smaller openings).

       Laboratory quality assurance samples and standards for the above methods.

       Markers, waterproof - for marking containers.

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

       pH buffers - Standard buffers at pH's of 4, 7, and 10 (or as per instructions or instrument manu-
              facturer) for standards and calibration check (see EPA, 2002a).

       Reagent water - defined as distilled or deionized water that does not contain substances which
              are toxic to the test organisms. A water purification system may be used to generate
              reagent water.

       Reagents for hardness and alkalinity tests (see EPA, 2002a).

       Reference toxicant solutions. Reference toxicants such as sodium  chloride (NaCl), potassium
              chloride (KCI), cadmium chloride (CdCl2), copper sulfate (GuS04), 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.

       Specific conductivity standards (see EPA, 2002a).

       Tape, colored - for labeling test vessels.

       Vials, marked - for preserving specimens for  verification.

       Yeast, cereal, and trout food (YCT) or equivalent substitute food - for feeding the cultures
              and test organisms.
B-l

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'i',J|, •; U.S. ENVIRONMENTAL PROTECTION AGENCY                    Ceriodaphnia Survival and Reproduction Toxicity Tests
^—•*                                                                        Supplement to Training Video
Appendix C

      APPARATUS AND EQUIPMENT LIST

       Balance -Analytical, capable of accurately weighing 0.00001 g.

       Bulb-thermograph or electronic-chart type thermometers - for continuously recording tem-
              perature.

       Counting Method for Algae - Electronic particle counter, microscope and hemocytometer,
              fiuorometer, or spectrophotometerto measure cell density (cells/mL) in the algal food
              concentrate.

       Disposable polyethylene pipets, droppers, and glass tubing with fire-polished edges,
              > 2-mm inner diameter, for transferring organisms.

       Dissecting microscope - for examining and counting the neonates in the test chambers.

       Environmental chamber, incubator, or equivalent facility with temperature control
              (25 ±  1°G).

       Filtering apparatus - for membrane and/or glass fiber niters.

       Glass or electronic thermometers - for measuring water temperatures.

       Light box - for illuminating organisms during examination.

       Light meter - with a range of 0 - 200 ^,E/m2/s (0-1000 ft-c).

       Mechanical shaker or magnetic stir plates

       National Bureau of Standards Certified thermometer (see EPA, 2002a).

       pH, DO, and specific conductivity meters - for routine physical and chemical measurements.

       Pipets bulbs and fillers - Propipet®, or equivalent.

       Pipettors, adjustable volume repeating dispensers - for feeding. Pipettors such as the Gibson
              REPETMAN®, Eppendorf®, Oxford®, or equivalent, provide a rapid and accurate means
              of dispensing small volumes (0.1 mL) of food to large numbers of test chambers.

       Racks for test vessels - Racks approximately 8 cm x 40 cm, drilled to hold 10 test vessels for
              each concentration.

       Randomization  Templates - Used for randomizing the placement of test concentrations for
              blocked randomization. Templates are prepared from poster paper and covered in plas-
              tic, made by laboratory

       Reference weights, Class S - for checking performance of balance. Weights should bracket the
              expected weight of the material to be weighed.

       Sample containers - for sample shipment and storage.

       Samplers - Automatic sampler, preferably with sample cooling capability, capable of collecting a
              24 hour composite sample of 5 L or more.

                                                                                          C-l

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 _£_ U.S. ENVIRONHENTAL PROTECTION AGENCY                     Ceriodaphnia Sur¥i¥al and Reproduction Toxicity Tests
                                                                               Supplement to Training Video
       Serological pipets - 1 - 10 mL, graduated.

       Test Chambers - 10 test chambers are required for each concentration and control. Test cham-
              bers such as 30-ml borosilicate glass beakers or disposable polystyrene cups are recom-
              mended because they will fit in the viewing field of most stereoscopic microscopes.

       Volumetric flasks and graduated cylinders - Class A, borosilicate glass or non-toxic plastic lab-
              ware, 10 - 1000 mL, for culture work and preparation of test solutions.

       Volumetric pipets - Class A, 1 - 100 mL.

       Wash bottles - for rinsing small glassware and instrument electrodes and probes.

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

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jS  U.S. ENVIRONMENTAL PROTECTION AGENCY                     Ceriodaphnia Sur¥i¥al and Reproduction Toxicity Tests
                                                                            Supplement to Training Video
                D

      FOOD AND ALGAE PREPARATION

      Feeding the proper amount of the right food is extremely important in Ceriodaphnia culturing
      and testing. The key is to provide sufficient nutrition to support normal reproduction without
      adding excess food. Excess food could clog the filtering apparatus of the organisms or greatly
      decrease the DO and subsequently cause decreased young production and possibly death of the
      animals. A suspension of yeast, cereal leaves, and trout food (YCT) along with the algae will pro-
      vide adequate nutrition when fed daily.

      The YCT is prepared from three ingredients as follows:
      Digested trout food or substitute flake food (Tetramin®, Bioril®, or equivalent), is prepared as
      follows:
          1.  Preparation of trout food or substitute flake food requires one week. Use starter or No. 1
             pellets prepared according to current U.S. Fish and Wildlife specifications (EPA, 2002a)

          2.  Add 5.0 g trout food to 1 L of MILLI-Q® water. Mix well in a blender and pour into a 2-L
             separatory funnel. Digest prior to use by aerating continuously from the bottom of the
             vessel for  one week at ambient laboratory temperature. Water lost due to evaporation is
             replaced during digestion. Because of the offensive odor usually produced during diges-
             tion, the vessel should be placed in a fume hood or other isolated, ventilated area.

          3.  At the end of digestion period, place in a refrigerator and allow to settle for a minimum
             of 1 hour.  Filter the supernatant through a fine mesh screen (i.e., Nytex® 110 mesh).
             Combine with equal volumes of supernatant from cereal leaves and yeast preparations
             (below). The supernatant can be used fresh or frozen until use. Discard the sediment.

      Cereal leaves (powdered or dried cereal leaves) are prepared  as follows:
          1.  Place 5.0 g of dried, powdered,  cereal or alfalfa leaves, or rabbit pellets,  in a blender.
             Cereal leaves or equivalent are available from commercial stores. Dried, powdered, or
             alfalfa leaves may be obtained from health food stores, and rabbit pellets are available at
             pet stores.

          2.  Add 1-L of MILLI-Q® water or equivalent.

          3.  Mix at high speed for five minutes in a blender or stir with a magnetic stirrer at medium
             overnight.

          4.  If blended, store in refrigerator overnight. If a magnetic stirrer is used,  allow to settle
             for 1 hour.

          5.  Decant the supernatant through a Nytex® 110 mesh screen and combine with equal
             volumes of supernatant from the trout food (above) and yeast preparations (below).
             Discard any excess.

      Yeast is prepared as follows:
          1.  Add 5.0 g of dry yeast to 1-L of MILLI-Q® water or equivalent.

          2.  Stir with a magnetic stirrer,  shake vigorously by hand or use a blender at low speed
             until the yeast is well dispersed.
                                                                                           D-I

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 _£_  U.S. ENVIRONHENTAL PROTECTION AGENCY                     Ceriodaphnia Sur¥i¥al and Reproduction Toxicity Tests
                                                                             Supplement to Training Video
           3.  Combine the yeast suspension immediately (do not allow to settle) with equal volumes
              of supernatant from the trout food and cereal leaf preparations (above). Discard the
              remainder.

       Combined foods as follows for the preparation of YCT:

           1.  Mix equal (300 mL) volumes of the three foods.

           2.  Measure suspended solids on each batch of YCT before feeding. Acceptable solids levels
              are between 1700 and 1900 mg/L. Record the date the food is prepared and the suspend-
              ed solids concentration.

           3.  Place aliquots of the final mixture in small (50 ml to 100 mL) screw-cap plastic bottles
              and freeze until needed, but no more than three months.

           4.  Fresh or thawed food is stored in the refrigerator between feedings, and is used for a
              maximum of two weeks.

       The algal concentrate is prepared as follows:
           1.  An  algal concentrate  containing 3.0 to 3.5 X 107 cells/mL is prepared from food cultures
              by centrifuging the algae with a plankton or bucket-type centrifuge, or by allowing the
              cultures to settle in a refrigerator for at least three weeks and siphoning off the superna-
              tant (See Chronic Methods Manual for description of food culture preparation).

           2.  The cell density (cells/mL) in the concentrate is measured with an electronic particle
              counter, microscope and hemocytometer, fluorometer, or spectrophotometer, and used to
              determine the dilution (or further concentration) required to achieve a final cell count of
              3.0  to 3.5 X lOVmL.

           3.  Assuming a cell density of approximately 1.5 X 106 cells/mL in the algal food cultures
              at 7 days, and 100% recovery in the concentration process, a 3-L, 7-10 day culture will
              provide 4.5 X 109 algal cells. This number  of cells would provide approximately 150 mL
              of algal cell concentrate (1500 feedings at 0.1 mL/feeding) for use as food. This would be
              enough algal food for four Ceriodaphnia dubia tests.

           4.  Algal concentrate may be stored in the refrigerator for two weeks.

       The quality of each batch of food prepared with a new supply of components should be deter-
       mined by using the food in a 7-day reproduction test with control water.
D-2

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   U.S. ENVIRONMENTAL PROTECTION AGENCY
Appendix E
      CERIODAPHNIA DUBIA ANATOMY
                                               Ceriodaphnia Survival and Reproduction Toxicity Tests
                                                                Supplement to Training Video
         Gut with Food
         Eggs in
         Brood Pouch
                                                    Swimming Legs
                                           — Female Claw
                                                                             E-I

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n
c

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WHOLE EFFLUENT TOXICITY • TRAINING VIDEO SERIES • Freshwater series
        Culturing of Fathead Minnows
        (Pimephales promelas)
        Supplement to Training Video

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                         NOTICE

The revision of this report has been funded wholly or in part by the
  Environmental Protection Agency under Contract EP-C-05-046.
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 of             (Pimephales promlas)
                                                                        Supplement to Training Video
  This report serves as a supplement to the video "Culturing of Fathead Minnows (Pimephales
  promelas)" (EPA, 2006a). The methods illustrated in the video and described in this report sup-
  port the methods published in the U.S. Environmental Protection Agency's (EPA's) Methods for
  Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms, Fifth Edition
  (2002a) and Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
  Waters to Freshwater Organisms, Fourth Edition (2002b), referred to as the Acute and Chronic
  Methods Manuals, respectively. The video and this report provide details on setting up and
  maintaining cultures based on  the expertise of the personnel at the EPA's Mid-Continent Ecology
  Division  (MED) in Duluth, Minnesota (EPA-Duluth). More information can also be found in
  Guidelines for the Culture of Fathead Minnows (Pimephales promelas} for Use in Toxicity Tests
  (EPA, 1987).

  This report and its accompanying video are part of a series of training videos produced by EPA's
  Office of  Wastewater Management. The video entitled "Fathead Minnow (Pimephales promelas)
  Larval Survival and Growth Toxicity Test" (EPA, 2006b) complements the material in this video
  by explaining the 7-day subchronic toxicity test method. These videos are available through the
  National  Service Center for Environmental Publications (NSCEP) at (800) 490-9198 or
  nscep@bps-lmit.com. Other freshwater videos include "Ceriodaphnia Survival and
  Reproduction Toxicity Tests" (EPA, 2006c).

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U.S. ENVIRONMENTAL PROTECTION AGENCY                             Culturing of Fathead Minnows (Pimephales promlas)
                                                                                            Supplement to Training Video
                                            Intentionally Left Blank

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U,S, ENVIRONMENTAL, PROTECTION AGENCY                    Culturing of Fathead Minnows (Pimephale* prom/as)
                                                                Supplement to Training Video
  Foreword	i

  Introduction	1

  Culture Water Preparation and Delivery	1

  Natural Water	2

  Dechlorinated Water	2

  Reconstituted Water	2

  Water Delivery	4

  Food Preparation	5

  Feeding The Larvae	5

  Live Brine Shrimp	5

  Frozen Brine Shrimp	5

  Supplements	6

  Cultures	6

  Initiating Cultures	6

  Breeding	6

  Spawning	7

  Collecting The Embryos	7

  Hatching	7

  Tracking The Fish	8

  References	11

  Glossary	Glossary-1

  Appendix A:  Illustration of Fathead Minnow with
               Anatomical Identifications	A-l

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e ; U.S. ENVIRONMENTAL PROTECTION AGENCY                           of                   prom/as)
                                                                 Supplement to Training Video
     TABLES

     Table 1. Recommended Chemical Characteristics of an
             Acceptable Culture Water	2

     Table 2. Water Quality Parameters for Reconstituted Waters	3

     Table 3. Preparation of Synthetic Fresh Water	4

     Table 4. Water Quality Parameters of Synthetic Freshwater
             Using Mineral Water	4
     FIGURES

     Figure I.Male Fathead Minnow and Female Fathead Minnow	6

     Figure 2.Suggested Daily Tasks for Fathead Culturing	8

     Figure 3.Testing Request Form	9

     Figure 4.Performance Tracking Form	10

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 JJ,  U.S. ENVIRONMENTAL PROTECTION AGENCY                       Culturing of Fathead Minnows (Pimephales promlas)
                                                                           Supplement to Training Video
Introduction
      Fathead minnows (Pimephales promelas) have been cultured for use in aquatic toxicity tests
      for over 30 years, and are the most common fish species used to determine sublethal toxicity of
      chemicals and complex effluents. The fathead minnow has a widespread distribution and is an
      important freshwater forage fish. It is also readily cultured in captivity. A large database on the
      effects of single chemicals has been developed using the fathead minnow for acute partial and
      life-cycle tests.
      Modifications to the 32-day early life stages (ELS) test produced a 7-day larval growth and sur-
      vival freshwater toxicity test. Norberg and Mount (1985) described this rapid method to assess
      the chronic toxicity of effluents using fathead minnows.

      Healthy animals are the most important aspect for a good toxicity test. Emphasis should be
      placed on determining the quality of the organisms used for producing the test organisms. This
      report and the video Culturing of Fathead Minnows (Pimephales promelas} were produced by
      EPA to clarify and expand on culturing methods explained in the Acute Methods Manual (EPA,
      2002a). To ensure successful toxicity testing, laboratory personnel should be familiar with the
      handling and culturing procedures detailed below.

      The fathead minnow is an adaptable organism and can be cultured in the laboratory under a
      variety of conditions. Factors that must not be overlooked are the types of food to use for lar-
      vae and adults, the stocking rates to grow testing and breeding stock, and the water to use. For
      example, use of the brine shrimp (Artemia) as a basic food source has been essential at EPA-
      Duluth. Dried or processed fish foods such as the Purina Aquamax® trout food (formerly Trout
      Chow) can be very good as a dietary supplement, but few  cultures have been successful using
      those diets alone. For cultures that require constant reproduction, the use of the fresh or frozen
      Artemia has been essential.

      The density of the fish in the culture is very important. While fish can survive at high stocking
      densities, rapid growth, uniformity of size, sexual maturation, high reproduction rates, and lim-
      iting the spread of disease can be achieved with lower densities. With good diets, space, constant
      temperature and photoperiod, the fathead minnow can be cultured with ease. Keen observation
      and regular maintenance of the culture animals and conditions are essential for year-round
      organism production.

      The first section of this report covers the selection of the culture water and explains procedures
      for natural, dechlorinated, and synthetic culture water preparation. The second section dis-
      cusses food requirements and preparation. The third section explains procedures for initiating
      and maintaining fathead minnow cultures. The methods described in this report cover culturing
      requirements for fish for use in both acute and chronic tests, although the emphasis is primarily
      on generating animals for sublethal tests.

Culture Water  Preparation  and Delivery
      The waters to be used for culturing fathead minnows are any toxicity-free freshwater includ-
      ing natural water, drinking water,  or reconstituted water. The water source chosen for culturing
      may  not necessarily be the same type of water used for testing. However, whichever water is
      chosen for culturing or testing, it must be tested to ensure that good survival and reproduction
      of the organisms are possible and that consistency is achievable. Before any water is used, it
      should be tested for possible contamination by pesticides, heavy metals, major anions and cat-
      ions, total organic carbon, suspended solids, or any other  suspected contaminants (OECD,  2006).
      The quality of the water should meet the acceptable levels described in Table 1. The water qual-
      ity should ensure adequate survival, growth, and reproduction and it should be from a consistent
      source to provide constant quality during any given testing period.

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,.S. ' U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                         Culturing of Fathead Minnows {Pimephales promlas)
                                                                           Supplement to Training Video
                  Table I.  Recommended Chemical Characteristics of an Acceptable
                  Culture Water (OECD, 1989)
Substance
Participate matter
Total organic carbon
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus Pol/chlorinated biphenyls
Total organic chlorine
Concentration
< 20 mg/L
< 2 mg/L
< 1 Ug/L
< lOug/L
< SO ng/L
< SO ng/L
< 25 ng/L
      NATURAL WATER
      Natural water can be from a variety of sources such as a surface water (e.g., river, lake, or pond),
      well water, or spring water. Natural waters should be carbon and/or sand filtered and fine fil-
      tered as well (~5ju,m). When using natural water that has resident fish populations, an ultraviolet
      sterilizer or ultrafilter may need to be added after the roughing filters to remove any potential
      fish pathogens.

      DECHLORINATED WATER
      Drinking water (i.e., city or tap water) may be used provided that it has received adequate treat-
      ment, but it may require dechlorination. This can be accomplished either by aeration for 24 hours
      or by using a carbon filter to remove residual chlorine. Sodium thiosulfate also may be used but
      it may act as a reducing/chelating agent in the water. The addition of 3.6 mg/L of anhydrous
      sodium thiosulfate will reduce 1.0 mg/L of chlorine. For fathead minnows, the 96-hour LC50 of
      sodium thiosulfate is 7.3 g/L (EPA, 1991). Following dechlorination, total residual chlorine should
      not exceed 0.01 mg/L. Because thiosulfate can be toxic to test organisms, a control lacking thio-
      sulfate should be included for any toxicity tests utilizing thiosulfate-dechlorinated water (EPA,
      2002b). Following dechlorination, tap water should be passed through a deionizer and carbon
      filter to remove metals and organics, and to control hardness and alkalinity.

      RECONSTITUTED WATER
      Two types of synthetic water are frequently used in testing, but the volumes required for cul-
      turing fish limit their application for culture. This is not to say that they are not suitable, but
      that their preparation is labor intensive, and the body of knowledge based on culturing in syn-
      thetic waters is small. Two types of synthetic fresh dilution water can be prepared. One is made
      using reagent grade chemicals and the other using a commercial mineral water. Both recipes are
      described in the Chronic Manual (EPA, 2002b). Recipes for preparing 20 liters of moderately hard
      water are given below.

      The deionized water may be obtained from a Millipore Milli-Q®, QPak2® or equivalent system.
      Acceptable ranges for the physical/chemical characteristics of the dilution water are provided
      in Table  2. In order to extend the life of the Milli-Q® cartridges, use a preconditioned (deionized)
      feed water by using a Gulligan®, Continental®, or equivalent system in front of the Millipore®
      system. In a four-cartridge Milli-Q® system place the cartridges in the order of (1) ion exchange,
      (2)  carbon, (3) organic cleanup such as Organex-Q®, and (4) a final bacteria filter (0.22-^,m fine fil-
      ter). For a five cartridge system, add an additional carbon cartridge. The order of the filter heads
      may need to be re-plumbed so that the water flows over the cartridges correctly. Conductivity
      of this filtered water should be zero ^,mhos/cm. All filters should be changed at least every six
      months, but more frequent changes may be needed. The frequency of change is dependent on the
      source water.

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C  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                          Culturing of Fathead Minnows (Pimephales promlas)
                                                                             Supplement to Training Video
                   Table 2. Water Quality Parameters for Reconstituted Waters
                   (EPA, 2002b)
Water Type
Very Soft
Soft
Moderately Hard
Hard
Very Hard
pH
6.4-6.8
7.2 - 7.6
7.4 - 7.8
7.6 - 8.0
8.0 - 8.4
Hardness*
10- 13
40-48
80- 100
160- 180
280 - 320
Alkalinity*
10- 13
30-35
57-64
110- 120
225 - 245
                   *Exf>ressed as mgll as CoC03

     The synthetic water made with reagent grade chemicals (Table 3) can be prepared in batches of
     20 L using the following recipe:
          •   Place 19 L of Milli-Q® or equivalent water in a properly cleaned plastic carboy.

          •   Addl.20gofMgS04, 1.92 g of NaHC03, and 0.080 g of KC1.

          •   Aerate overnight

          •   Add 1.20 g of CaS04»2H20 to 1 L of Milli-Q® or equivalent deionized water in a separate flask.
             Stir on a magnetic stirrer until the GaS04 is dissolved. Add to the 19 liters and mix well.

          •   Aerate vigorously for an additional 24 hours to dissolve the added chemicals and stabi-
             lize the medium.

          •   The measured pH, hardness, and alkalinity should be as listed in Table 2.

     The synthetic water prepared using commercially available mineral water also can be prepared
     in large batches (Table 3). The instructions in this report are specifically for Perrier® water (EPA,
     2002b). While other commercial waters have been tested, the properties of other waters have
     not been evaluated extensively; therefore no other commercial water instruction is provided. To
     prepare 20 L of water:
          •   Place 16 L of Milli-Q® or equivalent water in a properly cleaned carboy.

          •   Add 4 L of Perrier® water.

          •   Aerate vigorously for 24 hours to stabilize the medium.

          •   The measured pH, hardness, and alkalinity of the  aerated water should be as listed in
             Table 4.

          •   This synthetic water prepared with Perrier® water is referred to as diluted mineral
             water (BMW) in toxicity test methods.

     To aerate the water, use air free of oils and fumes. Organic vapors and oils can be removed using
     an in-line activated carbon filter such as Balston® C-l  (Balston, Inc., Lexington, MA.). Particles
     are removed using another in-line filter such as the  Balston® Grade RX filter, used frequently in
     combination with the carbon filter.

     Store both types of water in the carboys in which they were prepared and use each batch for only
     14 days. Water should be stored away from direct light, and should be kept covered. Bacterial
     growth may occur in the water as it ages, which can cause problems for the culture organism.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                       Culturing of Fathead Minnows (Pimephales promlas)
                                                                         Supplement to Training Video
    Table 3. Preparation of Synthetic Fresh Water (EPA, 200213)=*
Type
Very Soft
Soft
Moderately
Hard
Hard
Very Hard
Reagent Recipes
Reagent Added (rngll)
NaHC03
12.0
48.0
96.0
192.0
384.0
CaS04«2H20
7.5
30.0
60.0
120.0
240.0
MgS02
7.5
30.0
60.0
120.0
240.0
KCI
0.5
2.0
4.0
8.0
16.0
Mineral Water Recipes
Vol. of Mineral
Water Added (mLIL)
50
100
200
400
—
Proportion Mineral
Water (%)
2.5
10.0
20.0
40.0
—
    *Add reagent grade chemicals and lor mineral water to Milli-Q® or equivalent water.

                     Table 4. Water Quality Parameters of Synthetic
                     Freshwater Using Mineral Water (EPA, 2002b)
Water Type
Very Soft
Soft
Moderately Hard
Hard
Very Hard
pH
7.2-8.1
7.9 - 8.3
7.9 - 8.3
7.9 - 8.3
—
Hardness
10- 13
40-48
80- 100
160- 180
—
Alkalinity
10- 13
30-35
57-64
110- 120
—
  WATER DELIVERY
  If possible, a flow-through system should be used for culturing. The water delivery system
  should provide at least three to four turnovers per day. Threaded polyvinyl chloride (PVG) pipe
  is the most widely used construction material but glass, stainless steel, or Teflon® can be used.
  Rubber, copper, brass, or plastics containing fillers, additives, stabilizers, or plasticizers which
  may cause toxicity, and therefore, should not be used. Glued PVG should be used for drainage
  only, as the glue can be toxic.

  Recirculating systems often consist of aquaria at table top level for the fish, and an aquarium
  on the floor that acts as a trickling filter for the drain water from the fish tanks. This filter can
  be made of any non-toxic, high surface area material like crushed coral, pea gravel, or specially
  designed plastic media for trickling filters. These are available through filtration suppliers.
  Nitrifying bacteria in these filters convert ammonia to nitrate when the system is in balance.
  Ammonia levels must be monitored closely in this type of system. Water can be pumped from a
  pump at the bottom of the filter up to a headbox above the  fish tanks, where it flows back into
  the tanks.

  Gulturing fathead minnows is also possible using a static system. Each tank should have either
  an under-the-gravel or external filtration system. Supplies for these types of systems are avail-
  able at hobby shops and aquarium supply houses. Every 2 to 3 days, renew the water by siphon-
  ing down at least 25% of the volume and adding new water. Use distilled water when replacing
  water lost due to evaporation to avoid concentrating the dissolved salts. An additional consider-
  ation is that larvae must be protected from being captured in the filtering system. A container
  made of fine mesh will allow water to flow through, while  protecting the larvae.

  For breeding, the aquariums can be divided into four chambers with stainless steel mesh. In
  flow-through and recirculating systems, each tank can be serviced by one water source, air
  stone, and drain.

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jS  U.S. ENVIRONMENTAL PROTECTION AGENCY                       Culturing of Fathead Minnows (Pimephales promlas)
                                                                           Supplement to Training Video

      Fathead minnows are fed different forms of food during their development. From hatching to
      approximately 30 days their main diet is live brine shrimp (Artemia salina), and generally after
      one month old they are weaned over to partially frozen brine shrimp. The amount fed to each
      tank should be adjusted for the number and size of the fish.

      FEEDING THE LARVAE
      Feed the larvae live brine shrimp twice each day, Monday through Friday, and once each day on
      weekends. Larvae require very small amounts of food during the first few days (days 1-5), but
      require increasing amounts on days 6-10. After 10 days, the feeding rate must increase substan-
      tially each day and is proportional to the number of fish maintained. Care must be taken not
      to overfeed during the first few days,  and not to underfeed in the later stages. Careful observa-
      tion is critical and waste food on the bottom of the tank indicates overfeeding. This decaying
      food will cause the dissolved oxygen (DO) levels to drop. Rapid consumption of all food right
      after feeding (i.e., all Artemia is consumed 5-10 minutes post feeding) indicates underfeeding.
      Shortage of food is also evident by wide size variability in 30-day old juvenile fish. The rotifers
      Brachionus spp.  are an alternative food for the first feedings of larval fathead minnows.

      Observe the amount of food left at the end of the day and adjust the feeding rate accordingly.
      Each day, siphon out any excess food as waste food will grow fungus that can trap the larvae.

      LIVE BRINE  SHRIMP
      The brine shrimp used for feeding the larvae and juvenile fish Is Artemia salina. Upon receiving
      brine shrimp cysts from a supplier,  date the containers and store them in a freezer to prolong
      their shelf life.

      Brine shrimp cysts are most easily hatched in containers with conical shaped bottoms or in sepa-
      ratory funnels. Typically hatching instructions are provided with the cysts. A common proce-
      dure is to make a 25 - 30 ppt salinity  medium using un-iodized salt, and bubbling the water with
      filtered air from the bottom to keep the  cysts circulating.

      At 25° - 28°G, the Artemia begin to hatch in 24 hours. Larval fathead minnows must be fed less
      than 24-hour post-hatch Artemia nauplii so that they are small enough for the larvae to ingest.
      At this age, the nauplii also have their highest nutritional value as their yolk sacks have not yet
      been depleted. This nauplii size requirement makes it necessary to start new Artemia cultures
      daily.

      To  collect Artemia for feeding, remove the air supply and  allow the unhatched cysts to settle to
      the bottom of the hatching jar (approximately 5 minutes).  The live shrimp will settle forming an
      orange layer at the bottom of the container with a brown layer of unhatched cysts below it. The
      empty shells of the hatched cysts will rise to the top. The live shrimp can be removed using a
      large-bore pipette or a siphon. Either a 50-mL  or 100-mL pipette (inverted) works well. Before
      feeding in static systems, or for static tests, rinse the Artemia with distilled, deionized, or cul-
      ture water to prevent salt buildup in the tanks.

      FROZEN BRINE SHRIMP
      Fish that are over 30 days old are fed  partially frozen brine shrimp twice daily, Monday through
      Friday, and once a day on weekends. For easier handling, allow the brine shrimp to  thaw slightly
      at room temperature (not completely). Each spawning pair should receive approximately %-V4
      teaspoon of the brine shrimp. As a general guide, feed each tank of fish the amount of food that
      can be consumed in about 10 - 20 minutes.

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                         Culturing of Fathead Minnows (Pimephales promlas)
                                                                           Supplement to Training Video
      Nutritional quality and contaminant levels vary widely between strain, year of harvest, location
      of harvest, and supplier ofArtemia. It is useful to get as much information as possible from the
      supplier concerning the nutritional quality and contamination or have the Artemia checked for
      these parameters as new batches are ordered.

      SUPPLEMENTS
      The periphyton that grows naturally in the tanks provides a good dietary supplement for the fish.
      In addition, flake food such as commercially available Tetramin® or Purina Aquamax® trout food
      (formerly Trout Chow) can be used as supplements.

      Again, contaminant levels and nutritional content can vary widely, and screening and analysis
      may be required to ascertain the suitability of the foods.
Cultures
      INITIATING CULTURES
      Fathead minnow cultures should be started with fish from a reliable source such as a commer-
      cial supplier or a research laboratory. The embryos or fish should be shipped by overnight mail
      in an oxygenated container that is packed in a cooler to minimize temperature fluctuations. Upon
      receipt, allow the water in the shipping container to acclimate in a water bath or use aquarium
      heaters for the temperature adjustment. Once acclimated, empty the containers of starter cul-
      tures into a pan, aerate, and maintain the temperature at 25°G. In 4 - 5 days the embryos will
      hatch, at which time the larvae should be moved to rearing tanks using a large-bore pipette. For
      an 8- to 10-gallon tank, the recommended stocking density is 200-250.

      Once embryos hatch, feed them Artemia salina nauplii that are less than 24 hours old. Feed the
      fish 2 to 3 times a day,  5 days a week, for at least the first 2 weeks.  On weekends, feeding only
      once each day has proven to be adequate.

      BREEDING
      When the fish are 30 days old, they are removed from rearing tanks to be grown out as brood
      stock. In a 50- to 70-gallon tank, 300 - 400 fish can be grown to maturity. For the brood  stock
      fish, wean them over to frozen brine shrimp. To hasten the maturation process, thin the  3-4
      month  old fish to 30 - 35 per 10-gallon tank. The
      addition of spawning tiles may also speed up the
      maturation process.
      Fathead minnows begin to show signs of matu-
      rity at three to four months of age (Figure 1). The
      male will develop an enlarged head with rows of
      tubercles across the snout. These are used to clean
      the underside of the spawning substrate on which
      the eggs are deposited. The male will also develop
      black coloration on his sides. The female is smaller
      than the male and will not have tubercles. She is
      an olivaceous color and when mature, exhibits an
      ovipositor.

      Spawning tiles can be made of clay tiles or 10
      cm-diameter PVG pipes cut into 7 - 10 cm long
      sections which are cut in half lengthwise to create
      a semicircular arch. The inner side, where the eggs
Figure I. Male Fathead Minnow (top)
and Female Fathead Minnow (bottom)

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_£_ U.S. ENVIRONHENTAL PROTECTION AGENCY                       Culturing of Fathead Minnows (Pimephales promfos)
                                                                           Supplement to Training Video
      will be deposited, is then roughened with sandpaper. When placed in the aquariums, the fish use
      the underside to deposit and fertilize the eggs. The rough surface helps the eggs to adhere to the
      substrate. In the wild, fathead minnows use the underside of submerged or floating objects.

      Two options can be used for separating the fish for spawning. The first option separates the fish
      into spawning pairs. This is easily accomplished by dividing a 10- or 15-gallon aquarium into
      quarters using stainless steel screens. Paired spawning reduces competition which allows for
      greater production and makes it possible to monitor the fecundity of each pair. Daily records of
      reproduction can be used to identify sterile or spawned-out fish, which can be replaced by a new
      pair to maintain a high production rate.

      A second breeding option is to place groups of mature adult fish in tanks with a female:male ratio
      of 8:3. Use four spawning substrates in an 8- to  10-gallon tank with approximately 20 fish. When
      the egg production rate in these tanks drops, replace the entire group of fish.

      SPAWNING
      The male cleans the underside of the spawning tile with his tubercles and draws the female
      underneath. The male directs her toward the tile where she releases the eggs. Fertilization of the
      eggs is external and the buoyant eggs stick to one another and adhere to the underside of the tile.
      Females release an average of 100 - 200 eggs per spawn, with larger females releasing 200 - 400
      eggs. Fathead minnows spawn approximately every 4-5 days, but can spawn as often as every
      2 days.

      Monitor the reproduction rate of each brood pair or group of adults. If no embryos are produced
      in a 3-week period, replace the pair or the entire group. Younger fish can be allotted longer peri-
      ods of time if they are just beginning to spawn.

      COLLECTING  THE EMBRYOS
      The substrates in each tank should be checked daily for embryos. In order not to disrupt the
      early morning spawning, check the tanks midmorning. To retrieve the tiles, use tongs that are
      dipped in boiling water between each tank to minimize any possible transfer of disease from one
      tank to another.

      If needed for toxicity testing, the embryos can be removed from the tile with a gentle, circular,
      rubbing motion while keeping the tile underwater to prevent premature hatching caused by the
      disruption. If the embryos are to be hatched directly from the tiles, transfer the tiles immedi-
      ately to the hatching system.

      HATCHING
      Two options for hatching the fathead minnow embryos are to remove the embryos from the
      tiles to aerated water in separatory funnels or to keep them on the tiles and hatch them in aer-
      ated water in larger pans.  The first option requires that the embryos be rolled off of the tiles
      and pipetted into a separatory funnel containing aerating culture water. After 2 days in this
      system the embryos are placed in a pan containing aerated culture water. They will hatch in
      another 2-3 days.

      The second option is to place the tiles directly into a pan containing aerating culture water in a
      holding pan. A white pan allows the larvae to be seen more easily. The tiles are placed on their
      sides covered with culture water, and aerated. At a temperature of 25°G, the embryos should
      hatch in 4 - 5 days.

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S  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                         Culturing of Fathead Minnows (Pimephales pmmlas)
                                                                           Supplement to Training Video
     The aeration of the water in the separatory funnel and around the tiles provides circulation and
     helps keep sediment and fungal spores from settling on the embryos. Check the embryos on the
     tiles on days 1 and 2 for fungus or lack of viability and remove any such embryos with tweezers.
     On days 3, 4, and 5, check the tiles but minimize any disturbances as it may cause early hatch-
     ing of the larvae. Embryos that appear cloudy should be removed and discard all of the eggs from
     any tile on which 50% or more  die. Figure 2 is a check list used at EPA-Duluth to track the daily
     tasks required for fathead minnow culturing.

     After each use, the tiles are disinfected in a chlorine bath for 1 hour, rinsed with tap water, neu-
     tralized with sodium thiosulfate for at least 10 minutes to remove residual chlorine, and finally,
     rinsed in culture water and allowed to air dry.

     Larvae for future brood stock should be the progeny of as many adult spawning fish as possible.
     A few larvae collected each month from many different spawning pairs will provide a broader
     gene pool than hundreds of larvae from one or two spawning pairs in one week.

     TRACKING THE FISH
     If the fathead minnows are used for toxicity testing there will be a need to anticipate the
     demand for the eggs or larvae. The number of eggs that are needed for testing will determine
     the number left to hatch for larval testing or to grow out for future brood stock. Figure 3 is an
     example of a request form used to anticipate the demand on the cultures. Figure 4 is a tracking
     form that EPA-Duluth uses to monitor the performance of their broods that are used for toxicity
     tests. It represents one method used to track the health of the cultures.
DAILY CHECK-LIST FOR FATHEAD CULTURE UNIT (Initial when done)
DATE
MORNING
Check temperatures
Check water flow to
all tanks
Feed adult fish
frozen brine shrimp
Feed larvae live
brine shrimp
Check tiles in pans
for bad eggs
Pull spawning tiles
and est. no. eggs
(10:30)
AFTERNOON
Set up larvae
Feed adult fish
frozen brine shrimp
Feed larvae live
brine shrimp
Check temperatures
before leaving

Notes:


























































































































































      Figure 2. Suggested Daily Tasks for Fathead Culturing

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C  • U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                     Cylturing of Fathead Minnows (Pimephale$ pmmla$)
                                                                                           Supplement to Training Video
EGGILARVE REQUEST FORM
Name









Date









Need
(eggs or larvae)









How Many









When









Remarks









       Figure 3. Testing Request Form

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e   U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                                     Culturing of Fathead Minnows (Pimephale$ pmmla$)
                                                                                          Supplement to Training Video
FATHEAD CULTURE UNIT: Fish Tracking Form
Date:
Researcher:
Age of Fish (Embryos,
Larvae?):
Number Taken:
Type of Test (Embryo-Larval,
7 day, etc.):
Test Conditions (Static,
Renewal, Flow-Through?)
Temperature:
Dilution Water:
Test Chemical:
Hatching Percentage
(Embryos):
Control Survival (%) (Reps.
Different?):
Abnormal Survival in Low
Concentrations?:
Deformities in Control Fish?:
Observations (i.e., Condition
of Fish, Test Conditions):














RETURN FORM TO FATHEAD CULTURE UNIT BEFORE INITIATING NEXT TEST!
       Figure 4. Performance Tracking Form

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S  U.S. ENVIRONMENTAL PROTECTION AGENCY                       Culturing of             (Pimephales promlas)
                                                                          Supplement to Training Video
      EPA, 1987. Guidelines for the Culture of Fathead Minnows (Pimephales promelas) for Use in
             Toxicity Tests. EPA/600/3-87/001. Duluth, MN.

      EPA, 1991. Methods for Aquatic Toxicity Identification Evaluations, Phase I Toxicity
             Characterization Procedures. Second Edition. EPA-600-6-91-003. Duluth, MN.

      EPA, 2002a. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
             Organisms. Fifth Edition. 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 Freshwater Organisms. Fourth Edition. Office of Water, Cincinnati, OH. EPA-
             821-R-02-013.

      EPA, 2006a. Culturing of Fathead Minnows (Pimephales promelas}, Training Video. EPA-833-C-
             06-001. December 2006.

      EPA, 2006b. Fathead Minnow (Pimephales promelas} Larval Survival and Growth Toxicity
             Tests, Training Video. EPA-833-C-06-001. December 2006.

      EPA, 2006c. Ceriodaphnia Survival and Reproduction Toxicity Tests, Training Video. EPA-833-
             C-06-001. December 2006.

      Norberg, T.J. and D.I. Mount,  1985. A new fathead minnow (Pimephales promelas} subchronic
             toxicity test. Environ. Toxicol. Chem. 4:1-711-718.

      Organization for Economic Cooperation and Development (OECD), 1989. Draft Standard of the
             Fathead Minnow (Pimephales promelas} Larval Survival and Growth Test. OECD, Italy.

      Organization for Economic Cooperation and Development (OECD), 2006. Draft Proposal for a
             New Guideline, Fish Embryo Toxicity (FET) Test. OECD Guideline for the Testing of
             Chemicals. May 30,  2006 (1st Version), Draft Guideline. OECD, Italy.

      References are available online at www.epa.gov/npdes.

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 _£ • U.S. ENVIRONMENTAL PROTECTION AGENCY                       Culturing of Fathead Minnows (Pimephales pmmlas)
                                                                            Supplement to Training Video
Glossary
       Artemio. The marine invertebrate (referred to as brine shrimp) used as the recommended food
              source for culturing fathead minnows. Brazilian or Columbian strains are preferred
              because the supplies are found to have low concentrations of chemical residues.

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

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

       Ovipositor. The tubular extension of the female pore in certain fishes  used to assist in
              depositing eggs.

       Pimephales promelas. Scientific name for the fathead minnow, a common freshwater vertebrate
              minnow of the family Cyprinidae which is widely distributed  east of the Rockies.

       Tubercles. Spongy protrusions  on the dorsal surface of the male fish, anterior to the dorsal fin;
              these are used by the male to clean the debris from spawning  substrate  and fertilized
              embryos.
Glossary-1

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    U.S. ENVIRONMENTAL PROTECTION AGENCY
                                                         Culturing of Fathead Minnows (Pimephales promlas)
                                                                          Supplement to Training Video
Appendix A:
      ILLUSTRATION OF FATHEAD MINNOW WITH ANATOMICAL IDENTIFICATIONS
                                — Fat Pad
  Predorsal scales smaller —i
  anteriorly than posteriorly;
  especially crowded just
  behind the head.
                                       I— Dorsal Fin origin above or slightly
                                         anterior to pelvic fin origin
                         " = " = = = = = = = = = = == = = = ==„ = = = „.„, ,= :i = I!!=':


                                                    '
  Nuptial
  Tubercles
                                   Mala
                                                     — Incomplete Lateral Line
                                       Female
                                                   Ovipositor
                                                                                         A-1

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if

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WHOLE EFFLUENT TOXICITY • TRAINING VIDEO SERIES • Freshwater series
       Fathead Minnow (Pimephales
       promelas) Larval Survival and
       Growth Toxicity Tests
       Supplement to Training Video

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                         NOTICE

The revision of this report has been funded wholly or in part by the
  Environmental Protection Agency under Contract EP-C-05-046.
Mention of trade names or commercial products does not constitute
           endorsement or recommendation for use.

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S  U.S. ENVIRONMENTAL PROTECTION AGENCY   Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxieity Tests
                                                                            Supplement to Training Video
     This report serves as a supplement to the video "Fathead Minnow (Pimephales promelas) Larval
     Survival and Growth Toxicity Tests" (EPA, 2006a). The methods illustrated in the video and
     described in this report 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 Freshwater Organisms, Fourth Edition (2002a) and Methods for Measuring
     the Acute Toxicity of Effluents to Freshwater and Marine Organisms, Fifth Edition (2002b),
     referred to as the Chronic and Acute Method Manuals, respectively. The video and this report
     provide details on initiating, renewing, and terminating tests based on the expertise of the per-
     sonnel at the EPA's Mid-Continent Ecology Division (MED) in Duluth, Minnesota (EPA-Duluth).

     This report and its accompanying video are part of a series of training videos produced by
     EPA's Office of Wastewater Management. The video entitled "Culturing of Fathead Minnows
     (Pimephales promelas)" (EPA, 2006b) complements the material in this video by explaining
     the method for culturing fathead minnows for use in toxicity tests. These videos are available
     through the National Service Center for Environmental Publications (NSCEP) at (800) 490-9198
     or nscep@bps-lmit.com. Other available freshwater videos include "Ceriodaphnia Survival and
     Reproduction Toxicity Tests" (EPA, 2006c).

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U.S. ENVIRONMENTAL PROTECTION AGENCY     Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                                                  Supplement to Training Video
                                               Intentionally Left Blank

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Foreword	i

Introduction	1

Background	1

Test Method	1

Effluent Sampling	1

Dilution Preparation	2

Routine Chemistries	2

Test Chambers	3

Test Organisms	3

Feeding	3

Renewal	3

Test Termination	4

Test Acceptability and Data Review	4

Other Procedural Considerations	5

Diluent Water	5

Test Organism Cultures	5

References	5

Glossary	Glossary-1

Appendix A: Apparatus & Equipment List	A-l

Appendix B: Reagents & Consumable Materials	B-l

Appendix C: Summary of Test Conditions and Test Acceptability Criteria	C-l

Appendix D: Illustration of Fathead Minnow with
            Anatomical Identifications	D-l

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 NVIRONMENTAL PROTECTION AGENCY                                      and     Toxicity
                                                                Supplement to Training Video
TABLE

Table 1. Monitoring Schedule.
Figure 1. Male Fathead Minnow and Female Fathead Minnow	3

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 O • U.S. ENVIRONMENTAL PROTECTION AGENCY
                                      Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                           Supplement to Training Video
Introduction

      This report accompanies the Environmental Protection Agency's video training for conduct-
      ing freshwater fathead minnow (Pimephales promelas) larval survival and growth toxicity
      tests (EPA, 2006a). The test method is found in Short-term Methods for Estimating the Chronic
      Toxicity of Effluents and Receiving Waters to Freshwater Organisms (EPA, 2002a). The test is
      adapted from methods developed by Teresa Norberg-King and Dr. Donald Mount of EPA's Mid-
      Continent Ecology Division (MED), Duluth, Minnesota (Norberg and Mount, 1985). The mate-
      rial presented in both the video and this report 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 tox-
      icity. These permit limitations regulate pollutant discharges by a whole effluent toxicity (WET)
      approach rather than on a chemical specific basis.
  The test method requires a static
  renewal exposure system. Every 24
  hours, the fish are moved to a new tank
  containing a freshly prepared solution of
  the appropriate effluent concentration.
                                      The fathead minnow subchronic test is a freshwater seven-
                                      day static renewal exposure for determining sublethal toxic-
                                      ity in order to estimate toxicity. The test method determines
                                      the toxicity  of an effluent by exposing larval fathead min-
                                      nows (Pimephales promelas} to a series of effluent concentra-
                                      tions. The effect of the effluent is measured by the survival
                                      and growth  of the larvae. Minnows that are  24 hours old or
                                      less are exposed, and growth is measured as the difference
      in the larvaes average mean dry weight compared to that of the controls. This report covers the
      procedures for conducting the seven-day fathead minnow test and also describes some helpful
      procedures that are not presented in the Chronic Methods Manual.

Test  Method

      EFFLUENT SAMPLING
      Effluent sampling must be conducted according to the Chronic Methods Manual (EPA, 2002a) and
      any specific permit conditions. Samples are collected over a 24-hour period or when a 24-hour
      composite sampling period is completed. The time lapsed (holding time) from sample collection
      completion to first use of each grab or composite sample must not exceed 36 hours for test results
      to be acceptable for use in NPDES permit compliance testing. However, for all other testing pur-
                                      poses, no more than 72 hours should elapse between collec-
                                      tion completion and first use of the sample. In static renewal
                                      tests, each grab or composite sample also may be used to
                                      prepare test solutions for renewal at 24, 48, and/or 72 hours
                                      after first use if stored at 0° - 6°C, with minimum head space.
                                      Also according to the 2002 promulgated methods,  for WET
                                      samples with a specified storage temperature of 4°C, stor-
                                      age at a temperature above the freezing point of water to 6°C
      shall be acceptable (0° - 6°C). EPA has further clarified that hand-delivered samples used on the
      day of collection do not need to be cooled to  0° - 6°C prior to test initiation. (EPA, 2002c).
  Section 8 of the Chronic Manual covers
  sample collection. Note that surface
  waters should be filtered (60 ym
  plankton net) for fathead minnow tests.

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U.S. ENVIRONMENTAL PROTECTION AGENCY
                                   Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                          Supplement to Training Video
  DILUTION PREPARATION
  To start a test, warm the effluent to 25° ± 2°C slowly to avoid exceeding the desired tempera-
  ture. This is done using a water bath and monitoring the temperature closely. A temperature of
  25° ± 1°C should be maintained throughout the 7-day test period and the instantaneous tempera-
  ture must not deviate by more than 3°G during the test.

  Once the effluent and the dilution water have reached the desired temperature, the dilutions can
  be prepared. Use a minimum of five exposure concentrations and a control with a minimum of
  four replicates per concentration. The 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.

  ROUTINE CHEMISTRIES
              Once the various concentrations are prepared, set aside  one aliquot of each for the
              routine chemistries that must be performed. By setting these aside, the chemistries
              can be performed without contaminating 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 aeration is required, aerate all concentra-
              tions and the control. Take care not to cause excess turbulence that can cause physi-
              cal stress to the organisms.
                                   Dissolved oxygen, temperature, pH, and total residual chlo-
                                   rine must be measured on each new sample. EPA also recom-
                                   mends that total alkalinity, total hardness, and conductivity
                                   be measured on each new sample. Dissolved oxygen, tem-
                                   perature, and pH are 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 conductivity at
                                   the beginning and end of each 24 hour renewal is preferred
  but not required. The temperature and pH of the effluent sample also must be measured each day
  before preparing the test solutions. See Table  1.

  Table  I.  Monitoring Schedule
It is recommended that temperature be
recorded continuously or observed and
recorded in at least two locations in the
environmental control system or the
samples during the test.
Parameter
Dissolved oxygen1-2
Temperature1-2-3
pH1-2-3
Conductivity1-2
Alkalinity1
Hardness1
Total Residual Chlorine1
/Monitoring Frequency
Each New Sample
X
X
X
X
X
X
X
24 hour Exposure Period
Beginning
X
X
X
X
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 Measured in the effluent sample each day before preparation of new test solutions.

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  U.S. ENVIRONMENTAL PROTECTION AGENCY
                                     Fathead Minnow (Pimephales prome/as) Larval Survival and Growth Toxicity Tests
                                                                          Supplement to Training Video
     TEST CHAMBERS

              15cm
                               The Chronic Methods Manual recommends that test chambers
                               should not be smaller than a 500 mL beaker, yet allows test cham-
                               bers to be 1 L, 500 mL, or 250 mL plastic cups or fabricated rect-
                               angular (0.3 cm thick) glass chambers. The glass chambers should
                               measure 15 cm by 7.5 cm by 7.5 cm high. The test chambers should
                               be placed in a temperature and photoperiod controlled room or
                               environment and should be randomized after the test solution is
     added to each replicate. To avoid potential contamination
     from the air and excessive evaporation of test solu-
     tions during the test, the chambers should be covered
     with safety glass plates or sheet plastic (6 mm thick).
     Ambient laboratory lighting is sufficient for fathead
     minnow testing, but it should be on a controlled
     regime of 16 hours light and 8 hours dark. Ambient
     laboratory conditions are acceptable if they meet
     minimum environmental control standards and there
     are no large scale fluctuations.

     TEST ORGANISMS
     The test larvae should come from a pool of larvae con-
     sisting of at least three separate spawnings (Figure 1).
     To begin a test with five effluent concentrations and a
                            control, each with four repli-
                            cates, the minimum number
                            of larvae needed is 240. You
                            will need more than 240 to allow for extra larvae to choose from.
Calculation of Test Animals
5 effluent concentrations
+ 1 control
                                                          Figure I. Male Fathead Minnow (top)
                                                          and Female Fathead Minnow (bottom)
= 6 concentrations
x 4 replicates
= 24 tanks
x 10 animals/replicate
= 240 animals
     FEEDING
                            The larvae are placed one or two at a time into the test chambers until
                            each chamber contains ten larvae. To minimize the water volume add-
                            ed to each tank, the fish can be put in small beakers first. For example,
                            place one or two fish at a time in a small beaker until five are in each.
                            Then, reduce the water in each beaker to about 5 mL. Add these fish to
                            each tank until ten are in each replicate.
     Once the test is set up, the larvae are fed 0.1 g of concentrated Artemia nauplii three times per day
     or 0.15 g two times per day. The Artemia, or brine shrimp, should be started the day before test-
     ing begins. At 25°G, the brine shrimp will hatch in 16 to 18 hours. A fresh batch of brine shrimp
     should be prepared daily for the next day's use. Rinse the Artemia in freshwater and concentrate
     them in diluent water prior to each feeding. It is important that the larvae are fed 0.15 mL of the
     concentrate twice each day at least 6 hours apart to ensure live nauplii for the fish. Using less
     than 24-hour old. Artemia ensures a small size and provides the highest nutritional value.

     RENEWAL
     A fathead survival count must be recorded daily and all dead larvae removed. One method used
     to facilitate counting and cleaning is a light box which illuminates the larvae. During this phase
     of the test, take care not to disturb the larvae too much. The easiest method to remove the day-
     old effluent is to start a small siphon and lower the test media to a depth of 7 to 10 mm while
     removing all food particulates. That leaves approximately 15 to 20% of the total volume. An
     opaque Tygon® Y-tube cut off at an angle works well as a siphon, and the dark color causes the

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_£_ • U.S. ENVIRONMENTAL PROTECTION AGENCY    Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                            Supplement to Training Video
      larvae to move away. Another method is to use a large pipette, 50 to 100 mL capacity, fitted with
      a rubber bulb.

      Because of their small size, care must be taken not to remove any of the larvae. Collect the water
      as it is siphoned from the tanks in a white pan to facilitate observing any larvae that are inad-
      vertently siphoned from the chambers during cleaning. If a larvae is siphoned out and is still in
      good condition, transfer it back to the test tank. If a larvae is killed or injured, it should be duly
      noted and the larvae removed. This changes the initial number of fish in that replicate.

      To refill the tank pour the new test media slowly down the side of the test container. This will
      avoid excessive turbulence and prevent damage to the larvae.

      TEST TERMINATION
 Data Analysis:
 Complete data analysis procedures
 are presented in the appendices of
 the Chronic Methods Manual.
The larvae are not fed on day seven. A final survival count is made and the dead fish are
                           removed. The remaining fish can either be weighed immediately or
                           preserved in 70% alcohol for weighing later. It is extremely impor-
                           tant that the preserved larvae be weighed within two weeks of test
                           termination. To determine the final weights, first the weigh boats
                           are labeled; dried; and a tare weight measured. The fish are rinsed
                           with distilled water and all the fish from one replicate are placed
                           in one container. Dry the larvae at 100°G for at least 6 hours but
less than 24 hours. Weights should be obtained to the nearest 0.01 mg. After each group's weight
is determined, it is divided by the initial number of fish in that replicate. For the test to be
acceptable, control survival must be at least 80% and the control mean weight at least 0.25 mg.
The statistical analysis of the test results should be conducted according to the test manual.

TEST ACCEPTABILITY AND DATA REVIEW
Test data are reviewed to verify that test 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 dry weight per surviving organism in control chambers equal to or exceeding 0.25 mg.
However, the response used in the statistical analysis is mean weight per original organism for
each replicate, which is a combined survival and growth endpoint that is termed "biomass." Any
test not meeting the minimum TAG is considered invalid. All invalid tests must be repeated with
a newly-collected sample. Further guidance is provided in the Chronic Methods Manual.

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

EPA's promulgated toxicity testing method manuals (2002 a, b) recommend the use of point
estimation 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 con-
centration-response data,  and require calculating the percent minimum significant difference
(PMSD) when point estimation (e.g., LG50, IG25) analyses are not used. EPA specifies the PMSD
must be calculated when NPDES permits require sublethal hypothesis testing. EPA also requires
that variability criteria be applied as a test review step when NPDES permits require sublethal
hypothesis testing endpoints (i.e., no observed effect concentration [NOEG]  or lowest observed
effect concentration [LOEG]) and the effluent has been determined to have no toxicity at the
permitted receiving water concentration (EPA, 2002b). This reduces the within-test variability
and to increase statistical sensitivity when test endpoints are expressed using hypothesis testing
rather than the preferred point estimation techniques.

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 j& : U.S. ENVIRONMENTAL PROTECTION AGENCY   Fathead Minnow (Pimephales pmmelas) Larval Survival and Growth Toxicity Tests
                                                                             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.

       Acute toxicity test. A test to determine the concentration of effluent or ambient waters that
              causes an adverse effect (usually death) on a group of test organisms during a short-term
              exposure (e.g., 24, 48, or 96 hours). Acute toxicity data are analyzed using statistical
              procedures (e.g., point estimate techniques or a t-test).

       Artemia. The marine invertebrate (referred to as brine shrimp) used as the recommended food
              source. Brazilian or Columbian strains are preferred because the supplies are found to
              have low concentrations of chemical residues.

       Average mean dry weight. All the fish exposed in a given test chamber (replicate) are weighed
              together. The total dry weight is divided by the original number of 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.

       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 concentrations. Different dilutions, or concentrations, of an effluent used to determine
              the biological effects on test organisms (i.e., fathead minnows).

       Fathead minnow. Freshwater vertebrate fish species (Pimephalespromelas).

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

       LC50 (lethal concentration,  50%). The concentration  of toxicant or effluent that would  cause
              death to 50%  of the test organisms.

       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  Glean Water Act.

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

       Toxicity test. A procedure to determine the toxicity of a chemical or effluent using living
              organisms. A toxicity test measures the degree of effect of a specific chemical or effluent
              on exposed test organisms.

       WET (Whole Effluent Toxicity). The total toxic effect of an effluent measured directly with a
              toxicity test.
Glossary-1

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   t U.S. ENVIRONMENTAL PROTECTION AGENCY   Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                            Supplement to Training Video
Appendix A

      APPARATUS AND EQUIPMENT LIST
       Fathead minnow and brine shrimp culture units (see the Acute Methods Manual). This test
              requires 240-360 larvae. It is preferable to obtain larvae from an in-house fathead
              minnow culture unit. If it is not feasible to culture fish in-house, embryos or newly
              hatched larvae can be shipped in well oxygenated water in insulated containers.

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

       Sample containers. For sample shipment and storage.

       Environmental chamber or equivalent facility with temperature control (25° ± I °C).

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

       Balance. Analytical, capable of accurately weighing larvae to 0.0000 1 g.

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

       Borosilicate glass beakers or aquaria, or non-toxic disposable  plastic labware. A minimum
              of four 500-mL beakers or glass aquaria (7.6 cm wide x 16 cm long x 8.0 cm high) are
              required for each concentration and 1 control. Aquaria  can have a 7.4 x 7.0 cm piece of
              60 mesh stainless steel or Nytex® screen glued 2.5 cm in across one end. The surface to
              volume ratios in 500 ml beakers and the glass aquaria are approximately the same. To
              avoid potential contamination from the air, the chambers should be covered during the
              test. The Methods Manual recommends that test chambers should not be smaller than
              a 500 mL beaker, yet allows test chambers to be 1 L, 500 mL, or 250 mL plastic cups
              or fabricated rectangular (0.3 cm thick) glass chambers. The glass chambers should
              measure 15 cm by 7.5 cm by 7.5 cm high.

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

       Volumetric pipets. Class A, 1 - 100 mL

       Serological pipets. 1-10 mL, graduated.

       Pipet bulbs and fillers. Propipet®, or equivalent.

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

       Wash bottles. For rinsing small glassware and instrument electrodes and probes.

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

       Bulb-thermograph or electronic-chart type thermometers. For continuously recording
              temperature.

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

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

       Drying oven. 50° - 150°C range for drying larvae


                                                                                           A-1

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 jS ; U.S. ENVIRONMENTAL PROTECTION AGENCY   Fathead Minnow (Pimephales pmmelas) Larval Survival and Growth Toxicity Tests
                                                                            Supplement to Training Video
                 B

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

       Effluent, surface water, and dilution water.

       Reagents for hardness and alkalinity tests. (See EPA, 2002a).

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

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

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

       Specific conductivity standards. (EPA, 2002a).

       Reference toxicant solutions. Reference toxicants such as sodium chloride (NaCl), potassium
              chloride (KCI), cadmium chloride (CdCl2), copper sulfate (GuS04), sodium dodecyl sulfate
              (SDS), and potassium dichromate (K2Cr207), are suitable for use in the NPDES Program
              and other Agency programs requiring aquatic toxicity tests.

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

       Brine Shrimp (Anemia sp.) Cysts. (EPA, 2002b.) Although there are many commercial sources of
              brine shrimp eggs, the Brazilian or Columbian strains are preferred because the supplies
              examined have had low concentrations of chemical residues. Each new batch o£ Artemia
              cysts must be evaluated for nutritional suitability against known suitable reference
              cysts by performing  a side-by-side larval growth test. It is recommended that a sample
              of newly -hatched Artemia nauplii from each new batch of cysts be chemically analyzed
              to determine that the concentration of total organic chlorine does not exceed 0.15 ju,g/g
              wet weight or the total concentration of organochlorine pesticides plus PCBs does not
              exceed 0.30 ju,g/g wet weight. If those values are exceeded, the Artemia should not be
              used.

       Test organisms. Newly-hatched fathead minnow larvae (EPA, 2002b).
B-l

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 ?/> U.S. ENVIRONMENTAL PROTECTION AGENCY
    •*
Fathead Minnow (Pimepha/es prome/as) Larval Survival and Growth Toxicity Tests
                                              Supplement to Training Video
Appendix C
        Summary of Test Conditions and Test Acceptability Criteria for Fathead Minnow (Pimephales
        promelas) Larval Survival and Growth Toxicity Tests with Effluents and Receiving Waters (Test
        Method 1000.0)
Test type
Temperature (°C)
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal of test concentrations
Age of test organisms
Larvae/test chamber and control
Replicate chambers per
concentration
Feeding regime
Cleaning
Aeration
Dilution water
Effluent concentrations
Dilution factor
Test duration
Effects measured
Test acceptability
Sampling requirements
Sample volume required
Static renewal (required)
25° ± I°C (recommended)
Must not deviate more than 3°C during the test (required)
Ambient laboratory illumination (recommended)
10-20 uE/m2/s (SO - 100 ft-c)(ambient lab levels) (recommended)
16 hours light, 8 hours darkness (recommended)
500 ml beakers or glass aquaria (recommended minimum)
250 mL/replicate (recommended minimum)
Daily (required)
Newly hatched larvae; < 24 hours old. If shipped, not more than 48 hours old;
24-hour range in age (required)
10 larvae/chamber (required)
4 (required minimum)
On days 0-6, feed O.I g newly hatched (< 24-hour old) brine shrimp nauplii
three times daily at 4-hour intervals, or as a minimum, 0.15 g twice daily at
least 6-hour intervals (at the beginning of the work day and prior to renewal,
and at the end of the work day following renewal). Sufficient nauplii are added
to provide an excess, (recommended)
Siphon daily, immediately before test solution renewal, (required)
None, unless DO concentration falls below 4.0 mg/L. Rate not exceed 100
bubbles/minute, (recommended)
Uncontaminated source of receiving or other natural water, synthetic water
prepared using Millipore Milli-Q® or equivalent deionized water and reagent
grade chemicals, or DMW. (available options)
5 concentrations and a control (recommended minimum)
Receiving water: 100% receiving water (or a minimum of 5) 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 control; average dry weight per surviving organism
in control chambers > 0.25 mg. (required)
For on-site tests, samples collected daily, and used within 24 hours of the time
they are removed from the sampling device. For off-site tests, a minimum of 3
samples (e.g., collected on day 1, 3 and 5) with a maximum holding time of 36
hours before first use. (required)
2.5 L per day (recommended)
         Source: EPA 2002a. Chronic Methods Manual. For the purposes of reviewing WET test data submitted under NPDES permits, each test conditions listed
         above is identified as required or recommended. See Subsection 10.2 of the Chronic Manual for more information on test review). Additional require-
         ments may be provided in individual permits, such as specifying a given test condition where several options are given in these methods.
                                                                                                              c-i

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    U.S. ENVIRONMENTAL PROTECTION AGENCY   Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
                                                                              Supplement to Training Video
Appendix D:
       ILLUSTRATION OF FATHEAD MINNOW WITH ANATOMICAL IDENTIFICATIONS
                                 ,— Fat Pad
  Predorsal scales smaller  —i
  anteriorly than posteriorly;
  especially crowded just
  behind the head.
  Nuptial
  Tubercles
                                         I— Dorsal Fin origin above or slightly
                                           anterior to pelvic fin origin

                             =:;=I====:=;:=::!=:=!=;:==:::::;!;=;;:=:!
                                     Male
1— Incomplete Lateral Line
                                         Female
                                                      Ovipositor
D-l

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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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