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
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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
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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
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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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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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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
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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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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.
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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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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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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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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)
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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)
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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
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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)
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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)
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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
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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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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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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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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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Fathead Minnow (Pimepha/es prome/as) Larval Survival and Growth Toxicity Tests
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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.
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U.S. ENVIRONMENTAL PROTECTION AGENCY Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests
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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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