United States	Office of Research and	EPA 600 3-90 077
Fnvironmenta1 Protection	Development	Septombei 1990
Agency	Washington DC i>0460
SEPA Sperm Cell Test
Using the Sea Urchin
Arbacia	p
Supplemental Report for
Training Videotape

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Supplemental Report for the
Sperm Cell Tests Using the Sea Urchin
Arbacia punctulata
Training Videotape
U.S. Environmental Protection Agency
Center for Environmental Research Information
26 West Martin Luther King Drive
Cincinnati, Ohio 45268
U.S. Environmental Protection Agency
Environmental Research Laboratory
South Feny Road
Narragansett, RI 02882
This report has been reviewed by the U.S. Environmental Protection Agency and approved for
publication. Mention of trade names, products, or services is not, and should not be interpreted as
conveying official EPA approval, endorsement, or recommendation.
PROPERTY OF
ENVIRONMENTAL PROTECTION AGENCY

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TABLE OF CONTENTS
page
Introduction	1
Obtaining and Maintaining Sea Urchins	2
Obtaining Gametes	2
Making Stock Solutions	3
Beginning the Test	6
Evaluating the Test	8
References	9
Appendix A - Preparing Hypersaline Brine
Appendix B - Materials Needed for Testing
Appendix C - Summary of Test Conditions
Appendix D - Sample Data Sheets

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Sperm Cell Tests Using the Sea Urchin
Arbacia punctulata
INTRODUCTION
The U.S. Environmental Protection Agency's (EPA's)
Environmental Research Laboratory in Duluth, Minnesota,
has developed a series of freshwater toxicity tests to
evaluate effluent toxicity. The tests use freshwater fish,
invertebrates, and plant species as indicators of toxic
effects to lakes, streams, and rivers. The EPA
Environmental Research Laboratory in Narragansett,
Rhode Island (ERL-N), has adapted the freshwater toxicity
testing approach and developed new methods to perform
similar testing in the marine or estuarine environment.
This report summarizes methods developed at the
Narragansett lab for estimating the chronic toxicity of
marine or estuarine effluents and receiving waters on the
gametes of the sea urchin, Arbacia punctulata. Sperm cells
are exposed to effluents or receiving waters in a static
system for 1 hour, then sea urchin eggs are added for 20
minutes of static exposure. The test measures the effect
of exposure on fertilization. The methods described in this
report and demonstrated in the accompanying tape are
detailed in the EPA methods manual, "Short-term Tests
for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Marine and Estuarine Organisms"
(EPA/600/4-87/028).
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OBTAINING AND MAINTAINING SEA URCHINS
Adult sea urchins can be ordered from commercial
biological supply houses, or collected along the Atlantic
coast. Keep male and female animals in separate tanks; a
20-liter aerated tank can hold about 20 adults. Allow
filtered seawater to flow into the tanks at a rate of 5
L/minute. The temperature should be 12 to 18 C.
Sea urchins are fed kelp of the species Laminaria
gathered from Atlantic coastal waters or ordered from
commercial supply houses. Supply the urchins with ample
food, renewing the kelp each week and removing decaying
kelp as necessary. Healthy sea urchins will attach to kelp
or aquarium walls within hours - any unhealthy animals
should be removed and should not be used for testing.
Every 1 to 2 weeks, empty and clean the tanks.
A stock of at least 12 males and 12 females will be
needed for routine testing. If the animals will be used on
site, transport them separated by sex in separate or
partitioned coolers packed with wet kelp and paper towels.
On site, the sea urchins will be transferred into separate
10-gallon aquarium tanks with gravel-bed filtration. Even
with Filtration, be sure to change the water periodically to
maintain good water quality.
OBTAINING GAMETES
Eggs and sperm are obtained from healthy animals
collected from uncontaminated waters. Transfer the
animals into a shallow bowl filled with enough seawater to
just cover their shells. Four females should yield enough
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anus
suranal
plate
ocular plate
genital plate
genital pore
madreporite
madreporic
plate
Schematic of the aboral
surface ofArbacia vunctulata.
with spines partly removed to
show structure, especially the
genital pores.
eggs to test 5 test dilutions plus 1 control, with 3 or 4
replicates. Eggs are obtained from the female sea urchins
by electrical stimulation. Touch the shells close to the
genital pores with electrodes from a 12-volt transformer
for about 30 seconds. The red eggs will pool on the sea
urchin shell above the genital pores. Collect the eggs
from the shell using a 10-ml disposable syringe with a
blunted large-gauge needle so that it will rest on the shell
without puncturing it After collection, remove the needle
and empty the eggs into conical centrifuge tubes. All vials,
pipets, and pipet tips used in this test should be soaked in
seawater overnight. Keep the eggs at room temperature
until use, but not longer than a few hours.
Obtain sperm from' four male sea urchins. Again,
place the animals in a shallow bowl with their shells just
covered with control seawater. Like the females, the
males are induced to spawn by placing electrodes from a
12-volt transformer against their shells for 30 seconds.
Hie sperm appear white. Collect the concentrated sperm
that pools on top of the shell using a 1-, 2-, or 3-ml
syringe with a blunted large-gauge needle. Keep the
sperm sample on ice, and record the collection time. The
sperm are held on ice for 1 hour prior to exposure.
MAKING STOCK SOLUTIONS
In preparation for the actual exposure, the sperm and
eggs are diluted to constant concentrations with control
water. This ensures reproducibility in the test results. In
Narragansett, laboratory workers use a standard data sheet
for calculating and recording dilutions (sample data sheets
are in Appendix D). Later, during the exposure period,
2,500 sperm will be present for every 1 egg.
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After collection, the sperm should be in a volume of
about 0.5 to 1 ml of control water in the collecting syringe.
(Keep all sperm dilutions on ice.) This is called the
"sperm stock" solution. Perform a 50% serial dilution for
counting from this sample.
To do this, add 400 ul of the stock to 20 ml of
control seawater in a vial labeled "A." Gently mix the
solution by pipetting up and down with a 5 ml pipet, or by
inverting the vial several times. The dilution in this vial is
1:50. Take 10 ml from vial A and add to 10 ml of water
in the next vial, "B." After mixing solution B, take 10 ml
out and add it to a third vial, "C," containing 10 ml of
water. Vial C now contains a 1:200 dilution of the
original sperm sample. Repeat the last step once more
into a vial labeled "D." Discard 10 ml from vial D so that
all vials contain 10 ml.
Next, make a 1:2,000 sperm suspension for counting.
The sperm must be killed for counting, so add 10 ml of
10% glacial acetic acid in control water to vial C, the
1:200 dilution. Cap vial C, and mix by inversion. Add 1
ml from vial C to a vial labeled "E," which contains 4 ml
of control water, and mix thoroughly. Vial E now contains
1/2,000th of the number of sperm in the original sperm
sample. Now the concentration of the sperm stock can be
determined. Place an aliquot of dilution E onto a
hemacytometer for counting. Allow the dead sperm to
settle for about 15 minutes.
Meanwhile, prepare a standard egg dilution of 2,000
eggs/ml. To wash the eggs, remove the supernatant water
from the settled eggs. Add seawater and mix carefully by
inversion. Spin the vial in a tabletop centrifuge at the
lowest possible setting for 3 minutes to form a lightly
packed pellet. Wash the eggs twice more. Discard the
eggs and start again if the wash water appears red; this
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means that the eggs are lysing and are unsuitable for
testing.
After washing, transfer the washed eggs to a beaker
containing 200 ml of control water. This is called the "egg
test stock." Next, make a 1:10 dilution of the test stock
for counting. Mix the stock solution using gentle aeration
until the egg solution is homogeneous. The aeration
device used in Narragansett is a 3-pronged diffuser
attached by flexible tubing to an air pump. Cut the point
from a pipet tip to make sure the eggs will not be
damaged, then transfer 1 ml of egg solution to a vial
containing 9 ml of control water. Mix by inversion.
Transfer 1 ml of the egg solution to a Sedgewick-
Rafter counting chamber. Count the number of eggs
under a dissecting microscope at 25X magnification. Ten
times the number of eggs in that milliliter equals the
number of eggs/ml in the egg stock. The target
concentration is 2,000 eggs/ml. If less than 180 eggs were
counted, allow the eggs to settle in the beaker, remove the
proper volume of supernatant water according to the
formula:
200 - (# of eggs counted) = ml to remove
If more than 200 eggs are counted, add the proper volume
of water:
' 200 - (# of eggs counted) = ml to add
Verify the concentration by counting 1 ml of another 1:10
dilution of the adjusted stock solution. The count for the
final dilution should equal 200 +. 20 eggs/ml. If there are
four replicates, a test of five test concentrations with a
control will require at least 25 ml of egg test stock
solution at 2,000 eggs/ml.
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When the sperm have settled in the hemacytometer,
use a compound microsope to count the inner 400 squares
on each side of the chamber. At 100X magnification, the
sperm will appear as small specks in the chamber grid.
Average the counts from each side of the hemacytometer.
The resulting number times 10,000 is the number of
sperm/ml in vial E. In turn, that number times 2,000 is
the sperm concentration in the original sperm stock
(because vial E contains a 1:2,000 dilution of the sperm
stock solution). Calculate the sperm concentration in each
dilution vial, then choose one that is greater than or equal
to 5 x 10T sperm/ml. That vial is diluted to a final
concentration of 50 million (5 x 107) sperm/ml and used as
the sperm "test stock."
Only about 2.5 ml of sperm test stock solution are
needed for testing 5 test solutions and a control, with 3 or
4 replicates: Hold the test stock on ice until the test
begins, but no longer than 1 hour.
BEGINNING THE TEST
Store the effluent or receiving waters in a 4 C
incubator or refrigerater until the tests begin, but not
longer than 48 hours. Always maintain the salinity of the
test samples at greater than 26 ppt. To do this, effluent
samples may need to be adjusted using hypersaline brine.
A recipe for hypersaline brine is provided in Appendix A
Receiving water samples with greater than 26 ppt salinity
may be tested without further salinity adjustment. To test
a series of decreasing concentrations of effluent, use. a
dilution factor of 0.5. When starting with effluent at 0 ppt
salinity, ERL-N starts with 70 percent effluent. A table
for preparing the samples is in Appendix A
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In Narragansett, disposable glass vials are used as test
chambers. The vials are arranged in the partitioned
cardboard box in which they are packaged. Perform a
serial dilution from the highest concentration of effluent to
the lowest, starting with 10 ml of the highest
concentration. Move 5 ml from that vial to the next, and
so on, finally discarding 5 ml from the last viaL There
should also be at least 3 replicates of each concentration
and the control solution. Warm the samples to 20 C by
placing the vials on the laboratory bench if the ambient air
is warm enough, or in an environmental chamber, before
adding the sperm.
One hour after the sperm are collected,, add 100 ul of
the well-mixed sperm test stock to each test and control
vial. Cover the chambers, record the time, and leave the
chambers at room temperature for 1 hour.
Next, mix the egg test stock using gentle aeration and
add 1 ml of that test stock solution to each exposure vial.
When all of the vials contain eggs, lift the storage box and
move it in circles to "swirl" and mix the egg-sperm
suspension. Cover the chambers, record the time, and
incubate the eggs and sperm at room temperature for 20
minutes.
After 20 minutes, end the test and preserve the
samples by adding 2 ml of 10 percent formalin in seawater.
Again, record the time, then cap the vials. It is best to
evaluate the test immediately after the conclusion of the
test. The vials may also be saved at room temperature
until evaluation, but not longer than 24 hours.
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EVALUATING THE TEST
This test demonstrates the effluent or receiving water's
effect on sea urchin fertilization. To evaluate this,
exposed and control eggs are examined under a microscope
and the number of unfertilized eggs in each test chamber
is recorded.
Cut the point from a pipet tip. For each replicate,
transfer about 80 to 120 ul of the preserved eggs to a
multiple-chamber counting slide. If a Sedgewick-Rafter
counting chamber is used, transfer about 1 ml to the cell.
Using a compound microscope at 100X magnification,
count about 100 eggs. This should be done with adequate
ventilation, preferably under a hood, because formalin is
harmful when inhaled.
For each test chamber, record the total number of
eggs counted, and the number that were not fertilized.
Fertilized eggs are surrounded by a fertilization membrane,
while unfertilized eggs lack this membrane. Abnormal eggs
are not counted. For the test to be acceptable, the
control chambers should show more than 50 percent
fertilization. The methods manual, "Short-term Tests for
Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Marine and Estuarine Organisms," explains how
to analyze and interpret the results of the sea urchin test.
The sea urchin sperm cell test is currently used to
assess the potential toxic effects of complex chemical
mixtures on marine and estuarine organisms. Used in
conjunction with chemical-specific methods, this test can
provide a comprehensive and effective approach to
assessing the impact of complex effluents discharged to the
marine and estuarine environments.
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REFERENCES
Aquatic Toxicity Testing Seminar Manual. 1985. U.S. EPA Environmental Research
Laboratory, Narragansett, RI. ERL-N Contribution No. 796.
(Provides detailed descriptions of the methods used at Narragansett to evaluate the
toxicity of discharges to marine and estuarine waters. Served as the basis of a series of
seminars conducted by ERL-N personnel.)
Biomonitoring for Control of Toxicity in Effluent Discharges to the Marine Environment. 1989.
U.S. EPA Center for Environmental Research Information, Cincinnati, OH; U.S. EPA
Environmental Research Laboratory, Narragansett, RI. EPA/625/8-89/015.
(Describes the use of biomonitoring as an effective water quality-based approach to
controlling the toxicity of discharges to estuarine and marine waters. Covers regulatory
background, testing methods, and case studies.)
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Marine and Estuarine Organisms. 1987. Environmental Monitoring and Support Laboratory,
Cincinnati, OH. EPA/600/4-87/028.
(Describes methods, quality assurance, laboratory safety, facilities and equipment, data
analysis, report preparation, and organism culture and handling for six short-term tests to
estimate the chronic toxicity of effluents and receiving waters.)
Technical Support Document for Water Quality-based Toxics Control. 1985. U.S. EPA Office
of Water Enforcement and Permits, Washington, D.C.
(Provides guidance for each step in the water quality-based toxics control process, from
screening to compliance monitoring.)
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APPENDIX A
PREPARING HYPERSALINE BRINE
BACKGROUND
Salinity adjustments are a vital part of using marine and estuarine species for toxicity
testing. The majority of industrial and sewage treatment effluents entering marine and
estuarine waters contain little or no measurable salts. Therefore, the salinity of these effluents
must be adjusted before exposing estuarine or marine plants and animals to the solutions. In
addition, it is important to maintain constant salinity across all treatments throughout the test
for quality control. Finally, matching the test solutions' salinity to the expected receiving
water's salinity may require salinity adjustments. ERL-N uses hypersaline brine, prepared from
filtered natural seawater, to adjust exposure solution salinities. Note that commercially available
artificial sea salts have not been sufficiently tested, and therefore are not recommended for all
of the subchronic toxicity tests at this time.
Hypersaline brine has several advantages over artificial sea salts that make it more suitable
for use in toxicity testing. Concentrated brine derived from natural seawater. contains the
necessary trace metals, biogenic colloids, and some of the microbial components necessary for
adequate growth, survival, and/or reproduction of test organisms. It may be held for prolonged
periods without any apparent degradation. Brine may be added directly to the effluent to
increase the salinity, or may be used as control water by diluting to the desired salinity with
deionized water. The brine can be made from any high quality, filtered seawater supply
through simple heating and aerating.
GENERATING THE BRINE
The ideal container for making brine from natural seawater has a high surface-to-volume
ratio, is made of a non-corrosive material, and is easily cleaned. Shallow fiberglass tanks are
ideal.
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Collect high quality (and preferably high salinity) seawater on an incoming tide to minimize
the possibility of contamination. Special care should be used to prevent any toxic materials
from coming in contact with the seawater. The water should be filtered to at least 10 um
before placing into the brine tank. Thoroughly clean the tank, aeration supply tube, heater,
and any other materials that will be in direct contact with the brine before adding seawater to
the tank. Use a good quality biodegradable detergent, followed by several thorough deionized-
water rinses. Fill the tank with seawater, and slowly increase the temperature to 40 C If a
heater is immersed directly into the seawater, make sure that the heater components will not
corrode or leach any substances that would contaminate the brine. A thermostatically
controlled heat exchanger made from fiberglass works well.
Aeration prevents temperature stratification and increases the rate of evaporation. Use an
oil-free air compressor to prevent contamination. Evaporate the water for several days,
checking daily (or more or less often, depending on the volume being generated) to ensure that
the salinity does not exceed 100 o/oo and the temperature does not exceed 40 C. If these
changes are exceeded, irreversible changes in the brine's properties may occur. One such
change noted in original studies at ERL-N was a reduction in the alkalinity of seawater made
from brine with salinity greater than 100 o/oo, and a resulting reduction in the animals' general
health. Additional seawater may be added to the brine to produce the volume of brine desired.
When the desired volume and salinity of brine is prepared, filter the brine through a 10-
um filter and pump or pour it dirdctly into portable containers (5-gallon Cubitainers or
polycarbonate water cooler jugs are most suitable). Cap the containers, and record the
measured salinity and the date the brine was generated. Store the brine in the dark at room
temperature until used.
SALINITY ADJUSTMENTS USING HYPERSALINE BRINE
To calculate the volume of brine (Vb) to add to 0 o/oo sample to produce a solution at a
certain salinity (Sf), use this equation:
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Vb * sb = sf  vf
where Vb	=	volume of brine, ml
S	=	salinity of brine, o/oo
Sf	=	final salinity, o/oo
V,	=	final volume, ml (brine brought to this volume with 0 o/oo sample).
To calculate the volume of brine (Vb) required to raise the salinity of an effluent or
receiving water sample (S.) to a certain salinity (Sf), use this equation:
Vb = [(Vf - SO - (V.  s.)]/(sb - SO
where Vb	=	volume of brine, ml
Sb	=	salinity of brine, o/oo
V,	=	volume of sample, ml
S,	=	salinity of sample, o/oo
Sf	=	final salinity , o/oo
Vf	=	final volume, ml (final volume is combined brine and deionized water plus the
sample volume; percent original sample in the final sample = V/Vf * 100).
For the sea urchin test, start with an initial experimental concentration of 70 percent
effluent (70 ml effluent at 0 o/oo + 30 ml brine at 100 o/oo), and perform a 0.5 serial dilution
to make exposure concentrations of 35, 17.5, 8.8, and 4.4 percent effluent. The dilutions can
be prepared directly in the test vials.
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APPENDIX B
MATERIALS NEEDED FOR TESTING
Lab Supplies
Thermometer
Refractometer (salinometer)
Graduated cylinders, various sizes
1-liter volumetric flasks
Rat-bottomed glass dishes (about 20 cm diameter)
600- to 1000-ml glass beakers
Disposable gloves
Parafilm
Calibrated dispensing bottles (delivering 1 to 10 ml)
Pipettors: 5 ul, 50 ul,. 200 ul, 1000 ul, 5 ml
Disposable pipet tips (200 ul and 1000 ul, with about 3 mm sliced off the point)
Ice bucket with cover
Conical centrifuge tubes appropriate for centrifuge
Test tube rack
1- to 3-ml and 10-ml syringes with blunt-tipped needles (cut off the point)
Aeration device for suspending egg solution
Disposable scintillation vials
Manual two-place cell counter
Sedgwick-Rafter counting chamber
Neubauer hemacytometer
Data sheets (Appendix D)
Pasteur pipettes with pipette bulbs
Multi-chambered counting slides
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Lab Equipment
Incubator (capable of maintaining 20 C)
12-V transformer with steel electrodes
Centrifuge (capable of ambient, low-speed spin)
Vacuum pump, tubing, pasteur pipettes
Air pump (Silent Giant) or pressurized air source, with tubing
Fume hood
Dissecting microscope
Compound microscope
Solutions
Hypersaline brine
Deionized water
10% glacial acetic acid (reagent grade) in sea water
10% formalin in seawater
MATERIALS FOR MAINTAINING ORGANISMS
Sea urchins, Arbacia punctulata '(about 1 dozen of each sex)
kelp, Laminaria sp.
Standard salt water aquarium or Instant Ocean Aquarium (capable of maintaining sea water at
15 C) with appropriate filtration and aeration system
B-2

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APPENDIX C
SUMMARY OF TEST CONDITIONS
Test type:
Salinity:
Temperature:
Light source:
Irradiance:
Test solution volume:
Test chamber size:
Number of treaments
per test:
Number of
test organisms:
Number of eggs and
sperm cells per treatment:
Number of replicate
chambers per treatment:
Dilution water:
Dilution factor:
Test duration:
Effect measured:
static, non-renewal
30 o/oo  2o/oo
19 to 21 C
ambient laboratory light
about 10-20 uE m"2 s'1
5 ml
20-ml disposable, glass liquid scintillation vials, not
pre-cleaned
minimum of 5 effluent concentrations and one
control
pooled eggs from 4 females and pooled sperm from
4 males per test
about 2,000 eggs and 5,000,000 sperm per vial
4 (minimum of 3)
uncontaminated source of natural seawater;
deionized water mixed with hypersaline brine or
artificial sea salts
0.5
1 hour and 20 minutes
fertilization of sea urchin eggs
C-l

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APPENDIX D
DATA SHEETS

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SPERM CELL TOXICITY TEST
DATA SHEET I 1
TESTID:
PERFORMED BY:
SPERM SOLUTIONS
HEMACYTOMETER CT, E:	x 104 - spa 'E'
sperm concentrations: E x 40 - A -	SPM
E x 20 - B -	SPM
Ex 5 - D -	SPM
I
SOLUTION SELECTED FOR TEST (>-?5 x 107 SPM):
DILUTION: SPM/(5 x 10) -	DF
( (DF) X 10) - 10 -	+ SW, ML
EGG SOLUTIONS
INITIAL COUNT:	- VOLUME FOR FINAL EGG STOCK
COUNT ON FINAL EGG STOCK:	(200 X 10 EGGS/ML)
TEST STOCKS	,
SPERM STOCK:	(5 x 10 SPM)
VOLUME ADDED/TEST VIAL:	(100 UL)
EGG STOCK:	(2000/ML)
VOLUME ADDED/TEST VIAL	(1 ML)
TEST TIMES
SPERM COLLECTION:
SPERM ADDED:
EGGS ADDED:
FIXITIVB ADDED:
SAMPLES READ:
SALINITIES:

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SPERM CELL TOXICITY TEST RAW DATA
PROJECT:
SCONTROL3:
TESTCOM:
SAMPLE COMMENT:
SAMPNUM SAMPTYPE TREAT RCONC
REP
CTD
UNFERT
1
100

2
100

General Comments:
3
100

SAMPLE COMMENT:
SAMPNUM SAMPTYPE TREAT RCONC
REP
CTD
UNFERT
1
100

2
100

General Comments:
3
100


SAMPLE COMMENT:
SAMPNUM SAMPTYPE TREAT RCONC
REP
CTD
UNFERT
1
100

2
100

General Comments:
3
100


SAMPLE COMMENT:
SAMPNUM SAMPTYPE TREAT RCONC
REP
CTD
UNFERT
1
100

2
100

General Comments:
3
100

VERIFICATION:
TESTID:	STUDY:
SCONTROLl:	SCONTROL2
EFFl:	EFFCTLl:
EFF2:	EFFCTL2:
EFF3:	EFFCTL3:
'ft U.S. GOVERNMENT PRINTING OFFICE: 1990 - 748-159/20499

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