I
United States	Office of Water	E^SOS 3-39-COIa
Environmental Protection	(EN-336)	Feooiary 1989
Agency
Ceriodaphnia Survival
and Reproduction
Toxicity Tests
Supplemental Report for ||H|
Video Training Tape
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SUPPLEMENTAL REPORT FOR VIDEO TRAINING TAPE ON
CERIODAPHNIA SURVIVAL AND REPRODUCTION 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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CERIODAPHNIA SURVIVAL AND REPRODUCTION TOXICITY TEST
TABLE OF CONTENTS
INTRODUCTION	 I
BACKGROUND 	 1
TEST METHOD . 		 2
OTHER PROCEDURAL CONSIDERATIONS	 9
REFERENCES		13
GLOSSARY	14
APPARATUS AND EQUIPMENT 		APPENDIX A
REAGENTS AND CONSUMABLE MATERIALS. . . 	APPENDIX B
FOOD PREPARATION		APPENDIX C
RECOMMENDED TEST CONDITIONS FOR CERIODAPHNIA
SURVIVAL AND REPRODUCTION TEST 		APPENDIX D
DIAGRAM OF CERIODAPHNIA	APPENDIX E

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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 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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INTRODUCTION
This report accompanies the Environmental Protection Agency's video training tape for conduc-
ting Ceriodaphnia seven-day survival and reproduction toxicity tests. The test method is found
in Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Fre1-.'.water Organisms and is adapted from methods developed by Dr. Donald Mount and Teresa
Norberg-King of EPA's Environmental Research Laboratory, Duluth, Minnesota. The material
presented in both the tape 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
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 control pollutant discharges on a whole effluent
toxicity basis rather than by a chemical specific approach.
The Ceriodaphnia short-term chronic test estimates the toxicity
of an effluent by exposing groups of test organisms to different
concentrations of an effluent for seven days. The test results
are based on survival and reproduction of the organisms
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The test method requires a
static renewal exposure
system. Every 24 hours
the Ceriodaphnia are moved
to a new test cups,
containing a freshly
prepared solution of the
appropriate effluent
concentration.
Cultures produce Brood
animals that produce Test
animals.
5 effluent concentrations
+ 1 control
= 6 concentrations
x 10 replicates
= 60 replicates
x 1 brood animal/replicate
= 60 brood animals
using static renewal system. This report covers the general
procedures used for conducting seven-day Ceriodaphnia toxicity
tests. It describes how the test is set up, started, monitored,
renewed, and terminated, followed by suggestions on maintaining
healthy test organisms.
TEST METHOD
The first step is to set up Ceriodaphnia cultures to produce the
young, or neonates, for the test. Cultures must be started at
least two weeks before brood animals are needed, and their
survival records should be maintained. In order to obtain a
sufficient number of test animals that are less than 24 hours old
and within 12 hours of each other, it is advisable to have one
brood animal for each replicate that will be used in the test.
For example, a test with 5 concentrations and a control, with 10
replicates for each concentration, will need 60 brood animals.
Four days prior to starting a test, place each of the 60 brood
animals in a separate 30 ml beaker or plastic cup containing
15 ml of culture media. The brood animals chosen should be at
least seven days old and producing their third brood. Feed the
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animals daily and renew the culture media every other day. The
test requires 2 to 24 hour old test animals, or neonates. They
must be born within 12 hours of each other and originate from
the same in-house culture. Neonates that are born within 2 or 4
hours of each other are even more desirable.
Testing Animal Preparation
On the day of the test startup, remove any young from the
beakers or transfer the brood animals to new beakers early in
the morning. Collect the young born in the first two to four
hours, and every two to four hours thereafter, recording their
ages as they are collected. Once enough test animals are
obtained, they can be pooled and set aside. These can easily be
held until the end of the day to start the test. If a brood
animal is not producing 3 broods in 7 days, with 8 to 14 young
in the third brood, it may have been overcrowded, underfed, or
stressed in some way.
Section 8 of the Chronic
Methods Manual covers
sample collection. Note
that surface waters must
be filtered (80 um plankton
net) for Ceriodaphnia 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 + 1°C slowly to avoid exceeding the desired
temperature. Maintain this temperature throughout the seven-day
test period. Once the effluent and the dilution water have
reached the desired temperature, the dilutions can be prepared.
Unless otherwise specified, use five exposure concentrations and
a control for each test, with ten animals per concentration and
15 ml of medium per replicate.

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Routine Chemistries
It is recommended that the
temperature be recorded
continuously during the
test.
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 at the
beginning of each 24 hour renewal in each test concentration.
This is to ensure that the dilutions are not supersaturated or
below 40% saturation. If they are, they should be aerated gently
for a short period of time. Dissolved oxygen measurements
should also be performed at the end of each 24 hour 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 renewal period. See Table 1.
Once prepared, the solutions are poured into test cups starting
with the control and continuing to move up in concentration.
The exposure begins by randomly placing one neonate into each
test beaker. Using a glass dropper or pipette, remove a neonate
from the brood cup in a small amount of water and release the
animal under the surface of the water in the test cup.
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Table 1. Monitoring Schedule
Parameter
Monitorine Freauencv

Beginning of
End of

24-hr exposure
24-hr exposure
Dissolved Oxygen
X
X>
Temperature
X
X
PH
X
X
Conductivity
X

Alkalinity^
X
X
Hardness^
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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Replicates should be randomly placed in their test position and
covered to prevent evaporation as well as contamination from the
laboratory environment. Glass is recommended for the covers, as
plexiglass warps with moisture. The test trays are then placed in
a temperature and photoperiod controlled room.
Another method for conducting the test is by using a randomized
block design of treatment. This is done by randomly selecting 10
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. 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. When
using the randomized block designed, test chambers are
randomized once at the beginning of the test. If the
concentrations are arranged according to rows, the position must
randomized daily.
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Feeding
The test animals are to be fed the same diet that the cultures
are fed. At the time of the daily effluent renewal, 0.1 ml of a
YCT food mixture and 0.1 ml of algae are added to each 15 ml of
See Appendix C -- Food
test medium. The YCT food mixture is made of yeast, Cerophyll,
Preparation
and trout chow. The algae used is Selenastrum capricornutum.
In the 15 ml test volume, the number of algae cells should be
approximately 230,000 cells per ml, and the suspended solids level
of the food mixture should be about 12 to 13 mg/1.
The test is conducted at a daily temperature control of 25 + PC
due to possible effects of temperature on reproduction. The
photoperiod should be controlled at 16 hours light to 8 hours
dark. Also, the light intensity and quality should be at a
minimum of>standard ambient laboratory conditions. Once the
test is initiated, the renewals are performed in the same manner
on days two through six. To do the daily renewals, prepare the
test solutions, distribute the solutions to clean beakers or cups,
and add the food. 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 to throw
away 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.
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Record Ceriodaphnia survival and reproduction observations daily.
You can expect to obtain three broods from the control animals
during the seven day test period, given that the control water is
reliable water. The first brood is usually released on day three
or four, the second brood on day five, and the last brood on day
six or seven. When counting the young, organisms are observed
more readily if viewed with a black background or a low power
dissecting microscope. When using a microscope, a IS ml water
volume in the beaker or plastic cup allows viewing of the entire
water column without adjustments of the microscope. Using a
bottom light source can also be helpful.
In the absence of toxicants, adults typically will produce an
average of 15 to 35 vounp in 7 days. Some effluents or
toxicants may cause the young to develop in the brood pouch but
not be released or they may cause the young to be aborted.
Since the Ceriodaphnia develop rapidly, it is important to
terminate this test and make observations within ± 2 hours of 7
days from initiation of the exposure. For the test to be
considered acceptable, the control animals should produce an
average of 15 young per surviving female and have a survival
rate of at least 80% or better.
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Data Analysis
izta analysis procedures
are presented in the
appendices of the Chronic
Methods Manual.
At the end of the test, the data are analyzed for reduced young
production and survival. Because this test utilizes reproductive
success as a measure of sublethal toxicity, variance analyses may
be used to calculate the effect level. The LC50 may be
calculated for the 24 and 48 hour periods if mortality occurred
during these time periods. The statistical analysis of the test
results should be conducted according to the test manual.
OTHER PROCEDURAL CONSIDERATIONS
Food Preparation
Trout Chow
No. 1 trout food pellets
'U.S. FWS Specification
Diet SD9-30). Available
from Ziegler Bros., Inc.,
P.O. Box 95, Gardners, PA
17324; Glencoe Mills,
Glencoe, MN 55416; or
Murray Elevators, 118 West
4800 South, Murray, Utah.
In addition to strict adherence to the test protocol, there are
other factors that influence test results. The first is food
preparation (See Appendix C -- Food Preparation). It is
important that the yeast-Cerophyll®-trout chow mixture (YCT) is
similar in composition from week to week. To prepare the food,
follow the general guidelines in the chronic manual. The trout
chow is prepared first. At the Environmental Research Lab in
Duluth, a 1 gallon Nalgene® container is cut off and inverted.
Then, 5 grams of the 1/8" trout chow pellets are added to 1 liter
of distilled water. Filtered air is then applied through the
narrow opening of the bottle, and bubbled rapidly. This
fermentation is continued for one week, during which time care
is taken to replace any appreciable water loss, and not to allow
any settling of particulates. Once the trout chow has fermented,
the supernatant is 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.
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Cerophvll is available from
Ward's Natural Science
Establishment, P.O. Box
92912, Rochester, NY
14692. Cereal leaves from
Sigma Chemical Co., (P.O.
Box 14508, St. Louis, MO
6."T 78) have been used
successfully in place of
Cerophyll®.
Yeast
Dry yeast such as
Fleischmann's® or St. Regis®
is used.
Aleae
The Cerophyll® of the YCT should be made up only 24 hours in
advance. Put the Cerophyll® in the same diluent water as was
used for the trout chow fermentation and stir on a stir plate for
24 hours at a moderate rate. Decant and filter the Cerophyll®
through a Nytex® screen. Cerophyll® is a powder of dehydrated
cereal of grass leaves with Vitamin A, B2, C, and K.
The yeast for the YCT is mixed in the same diluent water as the
other food items. It should be made up on the same day that all
of the components are put together. Mix, shake, or stir the
yeast and add it to the other components while in suspension.
After all three components are ready, mix equal volumes together
and shake well. Measuring and adjusting suspended solids in the
YCT can help insure that the food level is similar from batch to
batch.
For culturing the algae, the Selenastrum media from the chronic
manual is recommended. Again, the cell count of the algae must
be monitored with each batch by using a Coulter Counter, a
hemacytometer, or by measuring the optical density. The algae
cells should be monitored occasionally to be sure that the culture
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is not contaminated with any other types of algae. Store both
foods in the refrigerator for no more than two weeks.
Culturing the test organisms is another factor that is important
for testing. Low production during the seven-day test may be
due to factors other than toxicity. 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. One consideration is to be
sure that the cultures are not overcrowded. If mass cultures are
used to produce test animals, it is highly likely that the animals
were subjected to stress due to underfeeding or overcrowding.
To minimize stress, maintain cultures with no more than 40
females per liter of water. Although mass cultures may appear
healthy, overcrowded females often produce fewer young than is
expected when used in a test. Generally, it takes an adult more
than four days to overcome these stresses. It is also important
that cultures are fed daily and that the mass cultures are
routinely changed, for example, on Monday, Wednesday, and
Friday, or the animals will quickly become overcrowded. A
general guideline that should be followed is to put as much effort
into culturing as into testing. The success of the test relies on
good culturing techniques.
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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 culture
performs, count young production for seven days each month for
ten animals in the culture 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 the following order
o ion exchange,
o carbon filter;
o organix-Q®; and
o fine filter.
Also avoid storing water for more than one month.
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 manual.
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REFERENCES
Short-term Methods for Estimating the 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.
Algae. The algae used is Selenastrum capricomutum.
Ceriodaphnia. Ceriodaphnia dubia are used for this test method.
Cerophyll®. A powder of dehydrated cereal of grass leaves used in the food
mixture.
Chronic toxicity. An adverse effect that occurs over a long exposure period.
The effect can be lethality, impaired growth, reduced reproduction, etc.
Diluent water. Dilution water used to prepare the effluent concentrations.
Effluent concentrations. Concentrations or dilutions of an effluent to which
fathead minnows are exposed to determine the effects of the sample.
Effluent sample. A representative collection of the discharge that is to be tested.
LC50. The toxicant concentration killing 50% of the exposed organisms at a
specific time of observation.
Neonate. Newly released Ceriodaphnia (less than 24 hours old) are used for this
test.
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 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.
Trout chow. No. 1 Trout Food pellets. U.S. FWS Specification Diet SD9-30.
Whole effluent toxicity. The aggregate toxic effect of an effluent measured
directly with a toxicity test.
Yeast. The yeast used is common dry yeast such as Fleischmann's® or St. Regis®.
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APPENDIX A
APPARATUS AND EQUIPMENT
Samplers -- Automatic sampler, preferably with sample cooling capability, capable
of collecting a 24-h composite sample of 1 L.
Sample containers - for sample shipment and storage.
Environmental chamber, incubator, or equivalent facility with temperature control
(25+ 1#C).
Water purification system -- Millipore Super-Q or equivalent.
Balance -- Analytical, capable of accurately weighing 0.0001 g.
Reference weights, Class S -- for checking performance of balance.
Racks for test vessels -- Racks approximately 8 cm x 40 cm, drilled to hold 10
test vessels for each concentration.
Dissecting microscope — for examining organisms in the test chambers.
Light box — for illuminating organisms during examination.
Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-
toxic plastic labware, 10-1000 ml, for culture work and preparation of test
solutions.
Volumetric pipets -- Class A, 1-100 ml.
Serological pipets -- 1-10 ml, graduated.
Pipets bulbs and fillers — Propipet®, or equivalent.
Disposable polyethylene pipets, droppers, and glass tubing with fire-polished edges,
2-mm ID -- for transferring organisms.
Wash bottles — 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.
National Bureau of Standards Certified thermometer — see EPA 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
which 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 -- Dilution water that contains
undesirable organisms, that may attack the test organisms should be filtered
through a fine mesh net (80-um or smaller openings).
Reagents for hardness and alkalinity tests (see EPA Methods 130.2 and 310.1,
USEPA 1979b).
pH buffers 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 EPA Method 120.1, USEPA 1979b).
Reference toxicant solutions.
Test Vessels -- 30-mL borosilicate glass beakers or disposable plastic salad
dressing cups (manufactured by Anchor-Hocking Plastic Division, and supplied by
Plastics Inc., 224 Ryan Avenue, St. Paul, Minnesota, 55164) are recommended
because they will fit in the viewing field of most stereoscopic microscopes. Rinse
thoroughly in distilled water and then in dilution water before use. A volume of
15 ml of test solution is adequate for the organisms, and will provide a depth in
which it is possible to count the animals with a minimum of re-focusing. Ten
test vessels are used for each effluent concentration and for the control.
Test Organisms -- Neonate Ceriodaphnia dubia 0-6 hours old of each other and
less than 24 hours old at the initiation of the test.
The Ceriodaphnia cultures should be started at least two weeks before the brood
animals are needed, to provide an adequate supply of neonates for the test.
Survival records on brood animals should be maintained. Only a few individuals
are needed to start a culture, since they are prolific.
Ceriodaphnia may be shipped or otherwise transported in polyethylene bottles.
Forty animals in a 1 L bottle filled with culture medium and smaller amounts
(3 ml) of the YCT diet is an acceptable way to transport. Organisms received
from an outside source should be immediately checked for temperature, and
transferred in water shipped into the laboratory culturing glassware or
plasticware. Animals received from an outside source should be transferred to
new culture media gradually, over a period of 1-2 days, to avoid mass mortality.
Keep the temperature change to <3°C over a 24 hour period.

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APPENDIX B CONTINUED
It is best to start the culture from one animal, which is sacrificed after producing
young, embedded, and retained as a permanent microscope slide mount to facilitate
identification and permit future reference. The species identification of the stock
culture should be verified by preparing slide mounts, regardless of the number of
animals used to start the culture. See the Methods Manual for slide preparation
procedures.
Glass dishes, beakers, or plastic cups are recommended for use in culturing. For
mass culture, use of crystallizing dishes, (190 x 100; 18.5 cm in diameter,
9.8 cm h) 1 L of water and 40 animals is recommended. Cultures are initiated
with <24 h old Q,. dubia. Smaller volumes of water using the same ratio of water
and animals is acceptable. Larger aquaria, i.e., 2 L, or 4 L, may be convenient,
but is not recommended for brood animal culture. Maintain several cultures in
separate vessels, (four or more) to provide additional cultures in case cultures are
accidentally dropped or lost due to other problems, such as low DO or lack of
feeding. The cultures are fed daily.
For individual organism culture, use of 1 ounce plastic cups or 30 ml glass
beakers with 15 ml of culture medium is recommended. One <24 hour old Q. dubia
per 15 ml is used. The number of animals needed in brood boards is dependent
on the number needed for tests. Typically, 60 animals of one group will produce
enough young to start one and possibly two test on one day.
Cultures should be changed three times a week, typically Monday, Wednesday and
Friday. Two sets of animals should be maintained, i.e., one culture is started
Friday, and the other culture started the previous Friday is 7-d old. The newest
culture is started with neonates <24 h old from the 14-d old culture (which is
then discarded). This same procedure is used for mass or individual cultures.
Daily changes are acceptable but not necessary.

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APPENDIX C
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, Cerophyll®, and trout chow (YCT) along with the algae,
Selenastrum capricornutum. will provide adequate nutrition when fed daily.
The YCT is prepared from three ingredients as follows:
Digested trout chow:
1.	One week before use, add 5.0 g of No. 1 trout food pellets (U.S. Fish
and Wildlife Service Specification Diet SD9-30) to 1 L of distilled or
deionized water or water used in culturing. This trout chow may be
obtained from Ziegler Bros., Inc., P.O. Box 95, Gardners, PA, 17324;
Glencoe Mills, Glencoe, MN 55416; or Murray Elevators, 118 West 4800
South, Murray, Utah. Mix well in a blender and aerate continuously
(digest) for one week at ambient laboratory temperature prior to use.
Be sure to aerate from the bottom to prevent settling, and replace water
lost to evaporation during fermentation.
2.	After one week of digesting, place in a refrigerator and allo.w the
mixture to settle. Decant the supernatant through a #110 Nytex® screen
and combine with equal volumes of supernatant from the Cerophyll® and
yeast preparations (below). The liquid can be frozen for use later.
Cerophyll® (Powdered, Dried, Cereal Leaves)' :
1.	One day prior to use, place 5.0 g of Cerophyll® powder in a flask. Add
500 ml of distilled, deionized water or water used for culturing. Mix at
high speed for five minutes in a blender or stir with a magnetic stirrer
at medium overnight.
2.	If blended, store in refrigerator overnight.
3.	Decant the supernatant through a #110 Nytex® screen and combine with
equal volumes of supernant from trout chow (above) and yeast
preparations (below). Discard any excess.
Available from Sigma Chemical Company, P.O. Box 14508, St. Louis,
Missouri, 63178 (800-325-3010).

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APPENDIX C CONTINUED
Yeast:
1.	Add 2.5 g of dry yeast, such as Fleischmann's® or St. Regis®, to 500 ml
of distilled water or deionized water, or water used for culturing.
2.	Stir with a magnetic stirrer or shake until well dispersed or use a
blender at low speed for 5 minutes. Do not allow to settle.
3.	Combine immediately with equal volumes of supernatant from the trout
chow and Cerophyll® preparations (above). Discard the remainder.
Combined trout chow-yeast-Cerophyll® food:
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/1. Record the
date the food is prepared and the suspended solids concentration.
3.	Place aliquots of the final mixture in small (50 ml to 100 ml) screw-cap
plastic bottles and freeze until needed.
4.	Fresh or thawed food is stored in the refrigerator between feedings, and
is used for a maximum of one week.
The quality of each batch of food prepared with a new supply of components
should be determined by using the food in a 7-day reproduction test with control
water.
Synthetic, soft or moderately hard water (hardness of 40-48 or 80-100 mg/1 as
CaCC>3) is recommended as a standard culture medium. Other culture water, such
as well water, pond water, or dechlorinated tap water, also may be satisfactory.
The Selenastrum is cultured as follows:
1.	Cultures can be purchased from Starr Collection at the University of
Texas, Austin, Texas or American Type Culture Collection, Rockville,
Maryland. Agar slants can be kept for several months in the dark at
4*C.
2.	Algae are grown using aseptic techniques although controlled bacterial
contamination does not appear to affect results.
3.	Algal media can be prepared in large quantities using several different
media. See the Chronic Methods Manual for details. Nutrient medium
is sterilized prior to adding algae by autoclaving or filtering through
0.22 um membrane filter.

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APPENDIX C CONTINUED
4.	Inoculate media aseptically with 2-5 ml of concentrated inoculum (see
below) or 30 ml of inoculum with optical density (O.D.) of 0.10-0.15 at
750 nm with 16.8 mm light path. Incubate cultures at 24 + 2°C and an
average of 2500 lux of illumination at the surface of flasks. Cultures
can be grown in Erlenmeyer flasks and magnetic .stirrers or separatory
flasks with air. If stir plates are used air temperatures of 21 + PC
will produce 24 + PC in culture.
5.	Cultures mature in 6-8 days at 25°C with a 16 hour light:8 hour dark
photoperiod. After cultures are very green this mixture can be
concentrated immediately or stored in the refrigerator until later. New
cultures can be started by adding 2-5 ml of the mature culture to
sterile media.
6.	Centrifuge mature algal cultures, pour off supernatant and resuspend
the algal pellet with diluent water, i.e., water used for culturing and
testing. Use only enough diluent to just break up pellet. Transfer all
resuspended algae into flask.
7.	Cell counts should be made using hemacytometers, coulter counters or
with spectrophotometers. The final concentration for £. dubia is
approximately 240,000 cells/ml. Therefore with more concentrated
algae, less volume is added to test solutions.
8.	Store algae in refrigerator. Shake vigorously before feeding. Algae
prepared in this manner stays viable for several weeks.
9.	Algae concentrated to 31 to 34x10** cells/ml should be used as stock.
10.	Vitamins can also be added to culture on day 2 if desired. Add 10 ml
to 1 L algal medium of the following:
Vitamins
mg/500 ml
D-pantothenic (Henri Calcium Salt)
Vitamin B-12 (Cyanocobalamin)
Thiamine HC1 (Vitamin B-l)
Riboflavin (Vitamin B-2)
Niacinamide (Nicotinamide)
d-Biotin (Vitamin H)
Putrescine (Dihydrochloride)
Myo-inositol
Choline chloride
12.0
8.0
26.0
6.0
6.0
220.0
100.0
140.0
0.006
These solutions should be stored in the dark in the refrigerator.

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APPENDIX D
RECOMMENDED TEST CONDITIONS FOR CERIODAPHNIA
SURVIVAL AND REPRODUCTION TEST
1.	Test type
2.	Temperature (°C):
3.	Light quality:
4.	Light intensity:
5.	Photoperiod:
6.	Test vessel size:
7.	Test solution volume:
8.	Renewal of test
concentrations:
9.	Age of test organisms:
10.	Number of test organisms
per chamber:
11.	Number of replicate chambers
per treatment
12. Feeding regime:
13.	Aeration:
14.	Dilution water
15. Test Solutions:
Static renewal
25 ± 1°C
Ambient laboratory illumination
10-20 uE/m^/s, or 50-100 ft-c
(ambient laboratory levels)
16 h light, 8 h darkness
30 ml
15 ml
Daily
Less than 24 h; and all released within a 6-h
period
I
10
Feed 0.1 ml each of the yeast-Cerophyll®-
trout chow food and the Seleastrum
capricornutum suspension/15ml exposure
chamber daily (algal suspension of 31,000,000-
34,000,000 cells/ml).
None
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.
Minimum of 5 effluent concentrations and a
control.
16. Dilution factor
Approximately 0.3 or 0.5

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APPENDIX D CONTINUED
17.	Test duration:
18.	Effects measured:
19.	Test acceptability:
20.	Sampling requirements:
7 days
Survival and reproduction
80% or greater survival, and an average of
>15 young/surviving female in the control
solution. At least 60% of control females
have produced their third brood.
Minimum of three 24-h composite samples.
For on-site tests, samples 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.
21. Minimum sample
volume daily:
500 ml

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APPENDIX E
DIAGRAM OF CERIODAPHNIA
8 9 10 12
5.
6.
7.
8.
9.
10.
11.
12.
13.
U.
15.
16.
17.
.18.
19.
3 2 1
Ancennule with ancerior sense hair and terminal aesthetascs (fine
Ocellus	sensory hairs)
r rons
Supraocular depression
Fornix
Fenestra (headpore)
Cervical notch
Cardiac bulge (hearc shown as dotted line beneath)
Ecdvsial line
Exopod of antenna
Endopod of antenna
Brood chamber
Abdominal appendage
Abdominal seta
Posterodorsal angle
Postabdomen
Anal denticles
Postabdominal claw with a pecten
Reticulations
EPA. 1986. Taxonomy of Ceriodaphnia (Crustacea; Cladocera) in U.S.
Environmental Protection Agency Cultures. September, 1986. EPA/600/4-86/032.
34 pp.

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