United States	Office of Water	EPA 505-8-89-00 >b
Environmental Protection.	(EN-336)	Feb'ruary 1989
'Agency
Fathead Minnow
Larval Survival
and Growth
Toxicity Tests
Supplemental Report For
Vid
eo Training Tape

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SUPPLEMENTAL REPORT FOR VIDEO TRAINING TAPE ON
FATHEAD MINNOW LARVAL SURVIVAL AND GROWTH TOXICITY TESTS
Lynn Bowler
Technical Resources, Inc.
3202 Tower Oaks Blvd.
Rockville, MD 20852
Laura Phillips
U.S. Environmental Protection Agency
Office of Water Enforcement and Permits
Permits Division
401 M Street, S.W.
Washington, D.C. 20460
Teresa Norberg-King
U.S. Environmental Protection Agency
Environmental Research Laboratory - Duluth
6201 Congdon Blvd.
Duluth, MN 55804
EPA Contract No. 68-03-3305
1988

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FOREWORD
The material presented in the video tape and this report is based on the
document Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. Some of the test conditions,
parameters, and methods of this manual are in the process of being revised and
were not published at the time of the completion of this project. The methods
presented here represent the latest accepted revisions.
This report has been funded wholly or in part by the Environmental Protection
Agency under contract 68-03-3305 to Technical Resources, Inc. It has been subject
to the Agency's review, and it has been approved for distribution as an EPA
document. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

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FATHEAD MINNOW LARVAL SURVIVAL AND
GROWTH TOXICITY TEST
TABLE OF CONTENTS
INTRODUCTION	1
BACKGROUND	1
TEST METHOD	2
OTHER PROCEDURAL CONSIDERATIONS	9
REFERENCES	10
GLOSSARY		11
APPARATUS AND EQUIPMENT. . 	APPENDIX A
REAGENTS AND CONSUMABLE MATERIALS		. APPENDIX B
RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
(PIMEPHALES PROMELAS') LARVAL SURVIVAL AND
GROWTH TEST	APPENDIX C

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INTRODUCTION
This report accompanies the Environmental Protection Agency's video training tape for
conducting fathead minnow larval survival and growth toxicity tests. The test method is found
in Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms. And is adapted from methods developed by Teresa Norberg-King and Dr.
Donald Mount of EPA's Environmental Research Laboratory, Duluth, Minnesota. The material
presented in both the videotape and this report summarzies the methods but does not replace a
thorough review and understanding of the methods by laboratory personnel before conducting the
test.
BACKGROUND
Clean Water Act,
Under the National Pollutant Discharge Elimination System
Section 402
(NPDES) program, 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 regulate pollutant discharges by a whole
effluent toxicity approach rather than on a chemical specific
basis.
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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 seven-day static renewal
exposure for determining sublethal toxicity in order to estimate
chronic toxicity. The test method determines the toxicity of an
effluent by exposing larval fathead minnows (Pimephales promelasl
to a series of effluent concentrations. The effect of the effluent
is measured by the survival and growth of the fish. Minnows
that are 24 hours old or less are exposed, and growth is
measured as the difference in the fishes' 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 manual.
TEST METHOD
Section 8 of the Chronic
Methods Manual covers
sample collection. Note
that surface waters must
be filtered (80 um plankton
net) for fathead minnow
tests.
Effluent sampling should be conducted according to the methods
manual. The test should be started on the arrival date of the
sample and within 72 hours of sample collection. Warm the
effluent to 25 + PC slowly to avoid exceeding the desired
temperature. This is done using a water bath and monitoring the
temperature closely. The temperature must be maintained
throughout the seven-day test period. 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 three or four replicates per
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concentration. The volume of test solution required is 250 ml
per replicate.
Routine Chemistries
It is recommended that the
temperature be recorded
continuously during the
test.
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 at the beginning of each 24 hour renewal in
each test concentration. This procedure will also be performed
at the end of the final exposure period for one replicate in each
concentration and the control. Also, the temperature, pH, and
conductivity must be measured and recorded at the beginning of
each exposure period. Temperature and pH must also be
measured and recorded at the end of each period. Alkalinity and
hardness are measured and recorded in the control and highest
concentration only, at the beginning and end of each 24 hour
period. See Table 1.
The test chamber should not be smaller than a one liter beaker
or a glass aquarium that is 7.6 cm wide by 16 cm long by 8 cm
high. The surface-to-volume ratio of the beaker and the
aquarium is approximately the same. 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.
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Table 1. Monitoring Schedule
Parameter
Monitoring Frequency
Beginning of
24-hr exposure
End of
24-hr exposure
Dissolved Oxygen
Temperature
PH
Conductivity
Alkalinity^
Hardness^
X
X
X
X
X
X
X
X
X
X
1 End measurement on one replicate in each concentration and the control.
^ Measure in highest concentration and control only.
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5 effluent concentrations
+ 1 control
= 6 concentrations
x 4 replicates
= 24 tanks
t 10 animals/replicate
= 240 animals
The test larvae should come from a pool of larvae consisting of
at least three separate spawnings. To begin a test with five
effluent concentrations and a control, each with four replicates,
the minimum number of larvae needed is 240. You will need
more than this to allow for extra larvae to choose from. The
larvae are placed one or two at a time into the test chambers
until each chamber contains ten larvae. To equalize the water
volume added to each tank, the fish can be put in small beakers
first. For example, place one or two at a time in a small beaker
until five are in each. Then, reduce the water in each beaker to
about S ml. Add these to each tank until ten fish are in each
replicate.
Feeding
Artemia are available from
Aquarium Products,
180 L. Penrod Ct.,
Glen Burnie, MD 21061.
Once the test is set up, the animals are fed 0.1 ml of
concentrated Artemia nauplii. The Artemia. or brine shrimp,
should be started the day before testing begins. At 25°C, 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 0.1 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.
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Ambient laboratory lighting is sufficient for fathead minnow
testing, but it should be on a controlled regime of sixteen hours
light and eight hours dark. Ambient laboratory conditions are
acceptable if they meet minimum environmental control standards
and there are no large scale fluctuations.
A fathead survival count should 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 ml
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 syphon, and the dark color causes
the fry 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, a white pan facilitates observing any fish that are
inadvertently siphoned from the chambers during the cleaning
operation. If a fish is siphoned out and is still in good
condition, transfer it back to the test tank. If an animal is
killed or injured, it should be duly noted and the animal removed.
This changes the initial number of fish in that replicate.

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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	The fish 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 important that the fish be
weighed within two weeks of test termination.
To determine the final weights, first the weigh boats are:
o labelled;
o dried; and
o weighed for tare weight
The fish are rinsed with distilled water and all the fish from one
replicate are placed in one container. Dry the fish at 100*C for
at least 2 hours but less than 24. Weights should be obtained to
the nearest 0.1 mg. After each group's weight is determined, it
is divided by the actual number of fish weighed. If any fish are
Data Analysis	unaccounted for in the weighing process, the group weight should
be divided by the number actually weighed—which could be
Data analysis procedures	different from the survival number. For the test to be
are presented in the	acceptable, control survival must be at least 80% and the control
appendices of the Chronic	mean weight at least 0.2S mg. The statistical analysis of the test
Methods Manual.	results should be conducted according to the. test manual.
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OTHER PROCEDURAL CONSIDERATIONS
Diluent Water
In addition to strict adherence to the test protocol, there are
other important factors that may influence test results. Two of
these are the choice of diluent water and the culturing of test
animals. The diluent water that is used is an important
consideration due to the fact that not all surface water is
reliable water for culturing. Therefore, before initiating a test,
it is important to establish the growth and survival rates are for
each water source. For artificially reconstituted waters, it is
very important to start with a "high purity" distilled and
deionized water. This may mean installing a high grade filtering
system and installing the filters in this order
o ion exchange;
o carbon filter;
o organix-Q®; and
o fine filter.
Also, avoid storing water for more than a month.
Methods for Measuring
Acute Toxicity of Effluents
to Freshwater and Marine
Organisms
Good cultures are important for ensuring reliable test results.
Culturing methods for fathead minnows are explained in the
Acute Methods Manual and is the subject of a separate training
tape.
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REFERENCES
¦	Short-term Methods for Estimating Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms. USEPA Office of Research and Development. December 1985.
EPA/600/4-85-014.
¦	Technical Support Document for Water Quality-based Toxics Control. USEPA Office of
Water. September 1985. EPA/440/4-85-032.
¦	Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms. USEPA Office of Research and Development. March 1985. EPA/660/4-85-013.
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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.
Artemia. Brine shrimp recommended as the 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 at one concentration are weighed together. The
total dry weight is divided by the number of fish weighed 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 the discharge that is to be tested.
Effluent concentrations. Fathead minnows are exposed to different dilutions, or concentrations,
of an effluent to determine the effects of the sample.
Fathead minnow. The species used is Pimephales promelas.
Larvae. The fathead minnow young are less than 24 hour old larvae at the start of the test.
LC50. The toxicant concentration killing 50% of the exposed organisms at a specific time of
observation.
Static renewal. The daily replacement of effluent medium in the test chamber.
Toxicity test. A measure of 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.
Whole effluent toxicity. The aggregate toxic effect of an effluent measured directly with a
toxicity test.
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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 150-300 newly hatched larvae. It is preferable to obtain these
fish from an inhouse fathead minnow culture unit. If it is not feasible to culture
fish inhouse, 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 4 L.
Sample containers — for sample shipment and storage.
Environmental chamber or equivalent facility with temperature control (25 + 1°C).
Water purification system — Millipore Super-Q or equivalent.
Balance -- analytical, capable of accurately weighing larvae to 0.0001 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.
Test chambers — borosilicate glass beakers or aquaria, or non-toxic disposable
plastic labware. A minimum of three 1-L beakers or glass aquaria (7.6 cm wide x
16 cm long x 8.0 cm high) are required for each concentration a^ji 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 1-L beakers and the glass
aquaria are approximately the same. To avoid potential contamination from the air,
the chambers should be covered during the test.
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 ID — for transferring
larvae.
Wash bottles — for washing embryos from substrates and containers and for rinsing
small glassware and instrument electrodes and probes.
Glass or electronic thermometers -- for measuring water temperatures.
Bulb-thermograph or electronic-chart type thermometers -- for continuously
recording temperature.

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appendix a continued
National Bureau of Standards certified thermometer (see USEPA Method 170.1,
USEPA 1979b).
pH, DO, and specific conductivity meters -- for routine physical and chemical
measurements. Unless the test is being conducted to specifically measure the effect
of one of the above parameters, a portable, field-grade instrument is acceptable.

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APPENDIX B
REAGENTS AND CONSUMABLE MATERIALS
Reagent water — defined as activated-carbon-filtered 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.
Effluent, surface water, and dilution water.
Reagents for hardness and alkalinity tests (see USEPA Methods 130.2 and 310.1,
USEPA 1979b).
pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for
standards and calibration check (see USEPA Method 150.1, USEPA 1979b).
Membranes and filling solutions for dissolved oxygen probe (see USEPA Method
360.1, USEPA 1979b), or reagents for modified Winkler analysis.
Laboratory quality assurance samples and standards for the above methods.
Specific conductivity standards (see USEPA Method 120.1, USEPA 1979b).
Reference toxicant solutions.
Alcohol (4%) for uses as a preservative for the fish larvae.
Brine Shrimp (Artemia) Cysts — see the Acute Methods Manual. 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. (One source is Aquarium Products, 180 L Penrod Ct., Glen
Burnie, MD, 21061). Each new batch of Artemia cysts should be evaluated for
nutritional suitability against known suitable reference cysts by performing a 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 0.1S ug/g wet weight or the
total concentration of organochlorine pesticides plus PCBs does not exceed 0.3 ug/g
wet weight. If those values are exceeded, the Artemia should not be used.
Limited quantities of reference Artemia cysts, information on commercial sources of
good quality Artemia cysts, and procedures for determining cysts suitability are
available from the Quality Assurance Branch, Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, 45268.
Test organisms -- Newly-hatched fathead minnow larvae (See Acute Methods
Manual).

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1.
2.
3.
4.
5.
6.
7.
8.
9.
10
11,
12.
13.
14.
15.
16
17
18
APPENDIX C
RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
PIMEPHALES PROMELAS't LARVAL SURVIVAL AND GROWTH TEST
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/
concentration:
Feeding regime:
Cleaning:
Aeration:
Dilution water
Effluent concentrations:
Dilution factor:
Static renewal
25 ± 1°C
Ambient laboratory illumination
10-20 uE/m^/s (50-100 ft-c)(ambient lab levels)
16 h light, 8 h darkness
1 -L beakers or glass aquaria
250 ml/replicate
Daily
Newly hatched larvae; <24 hours old
10 larvae/chamber; Minimum of 30 larvae/
test concentration
4 (minimum of 3)
Feed 0.1 ml < 24-hour old brine shrimp nauplii
in a concentrated suspension twice daily, at
least 6 hours between feedings
Siphon daily, immediately before test solution
renewal
None, unless DO concentration falls below 40%
saturation. Rate should be less than 100
bubbles/min.
Milli-Q or equivalent water is used to make a
standard moderately hard water, or diluted
mineral water (i.e 9 parts Milli-Q and 1 part
Perrier® or equivalent). Aerate a minimum of 12
hours before use.
At least 5 effluent concentrations and a control
Approximately 0.3 or 0.5
Test duration:
7 days

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appendix c continued
19.	Effects measured:
20.	Test acceptability
21.	Sampling requirements:
22.	Minimum sample volume
required daily:
Survival and growth (increase in weight)
80% or greater survival in control solutions and
a minimum weight of controls of at least
.250 mg.
Minimum of three 24-h composite samples. For
on-site tests, sample should be used within 24 h
of the time they are removed from the sampling
device. For off-site tests, samples should be
used within 36 h of collection.
2 L

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