£EPA
United States Environmental Monitoring Systems
Environmental Protection Laboratory
Agency Las Vegas NV 89114-5027
EPA/600/8-87/011
March 1987
Research and Development
User's Guide:
Procedures for
Conducting Daphnia
magna Toxicity
Bioassays
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EPA/600/8-87/011
March 1987
PROCEDURES FOR CONDUCTING DAPHNIA MAGNA TOXICITY BIOASSAYS
Prepared for the Office of Solid Waste
by
Kenneth E. Biesinger
Environmental Research Laboratory
Duluth, Minnesota 55804
and
Llewellyn R. Williams
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
and
William H. van der Schalie
U.S. Army Medical Bioengineering Research
Fort Detrick, Frederick, Maryland 21701-5010
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
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NOTICE
The Information in this document has been funded wholly or in part by
the United States Environmental Protection Agency under Interagency Agreement
DW21930057 to United States Army Research and Development Command. It has
been subject to the Agency's peer and administrative review, and it has been
approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation for
use.
ii
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FOREWORD
The procedures reported herein for conducting Daphnia magna toxicity
bioassays were successfully evaluated in a collaborative study involving eleven
government, industry, and academic laboratories with four toxicants. Results
of this study were reported at the annual meeting of the Society of Environmen-
tal Toxicology and Chemistry (SETAC) in November 1985 and were reported by
Williams et al., 1986.
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ABSTRACT
A standardized protocol has been developed to provide guidance for
conducting acute (death or immobility) and chronic (survival and reproduction)
toxicity of solid waste leachates to Daphnia magna. The method with slight
modifications is applicable for testing toxicants in general. Acute test
results are reported as a 48-hour IC$Q (concentration at which 50 percent
of test organisms are killed or immobilized after 48 hours of exposure) with
95 percent confidence intervals. Chronic test results are reported as 21-day
LCsos (concentration at which 50 percent of test organisms were killed during
21-day exposures) with 95 percent confidence limits, the lowest concentration
at which there was a significant (95 percent confidence interval) effect on
reproduction and the highest concentration at which there was no significant
effect.
iv
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CONTENTS
Foreword ................................ iii
Abstract ................................ 1v
Definitions .............................. yll
Acknowledgment ............................. viii
Summary ................................. 1
Culture and Testing Methods ....................... 2
General culture procedures for brood stocks ............ 2
Acclimation culture procedures ................... 2
Organisms ........................... 2
Food and feeding ....................... 3
Methods ............................ 3
Containers .......................... 3
Replication .......................... 3
Aeration ........................... 3
Cleaning ........................... 3
Light and phot ope Mod ..................... 4
Temperature .......................... 4
Water quality measurements .................. 4
pH .............................. 4
Acute Tests .............................. 5
Specific procedures ........................ 5
Organisms ........................... 5
Food and feeding ....................... 5
Methods ............................ 5
Containers .......................... 5
Leachates ........................... 5
Dilution water ........................ 5
Controls ........................... 5
Acetate controls ....................... 6
Test concentrations ...................... 6
Randomization ......................... 6
Replication .......................... 6
Aeration ........................... 6
Cleaning ........................... 6
Light and photoperiod ..................... 7
Temperature .......................... 7
Water quality measurements .................. 7
pH .............................. 7
Leachate measurements ................... • 7
Test apparatus ........................ 7
General acute test procedures ................... 7
Statistical evaluations ...................... 8
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CONTENTS (Continued)
Chronic static-renewal tests 9
Specific procedures 9
General chronic test procedures 12
Statistical evaluations 13
Survival 14
Reproduction and length 14
Confidence intervals and after-the-fact
power calculations 15
Obtaining and Recording Data 16
Acute 16
Chronic 16
Data Reporting 17
Literature Cited 19
References 21
Appendices
A. Reconstituted water preparation 22
B. Daphnia food preparation 24
C. Culturing Selenastrum capricornutum 25
D. Preparation of algae for feeding Daphnia 29
E. Equipment 31
F. Data forms 33
G. Quality assurance 40
H. Standard operating procedure for auditing Daphnia
acute EC test 42
VI
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DEFINITIONS
Acute toxicity: a relatively short-term lethal or other (e.g., immobiliza-
tion, equilibrium loss) effect, usually defined as occurring within 48
hours for Daphnia.
Chronic toxicity: full life-cycle (21 days for Daphnia) effects such as
changes in growth, reproduction, mutations, or death.
LC5Q: a statistically estimated toxicant concentration at which 50 percent of
exposed organisms would be killed at a specific time of observation; for
example, 48-hour, 7-day, 14-day, or 21-day LCsgs for Daphnia.
a statistically estimated toxicant concentration at which a specific
response (i.e., death or immobilization) would be elicited in 50 percent
of exposed organisms at a specific time of observation; for example,
48-hour EC5Q immobilization.
Immobilization: no visible movement of appendages when gently prodded.
Static bioassay: test in which solutions and test organisms are placed in
test chambers and kept there for the duration of test (24 or 48 hours
for Daphnia).
Renewal bioassay: a test with periodic exposure (Monday, Wednesday, and Friday
or a similar schedule) of test organisms to fresh test solutions of the
same composition. This is accomplished by transferring test organisms
into new test chambers containing the appropriate test solutions and food.
Trimmed Spearman-Karber Method: calculation method for median lethal or median
effect concentrations and 95 percent confidence intervals for toxicity data.
Dunnett's test: a multiple comparison of treatment means against the control
mean for analysis of variance.
Number of young: the total number of young that were produced during the test
period by those females that remained alive at the end of a chronic test.
Length: the total length (in mm, from the top of the head to base of the spine)
of those females that remained alive at the end of a chronic test.
VII
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ACKNOWLEDGMENT
The cooperation of the Review Panel in providing input to the development
and review of the interim test procedures is gratefully acknowledged.
Participants on the Review Panel include:
Dr. B. Adams, Monsanto Industrial Chemicals Co.
Dr. K. Biesinger, EPA/ERL-Duluth
Dr. A. Buikema, Virginia Polytechnical Institute
Mr. S. Ells, EPA/OTS Washington, D.C.
Dr. P. Feder, Battelle Columbus Laboratories
Dr. C. Goulden, Academy of Natural Sciences, Philadelphia
Dr. K. Keating, Rutgers University
Mr. T. Kimmel, EPA/OSW Washington, D.C.
Mr. G. LeBlanc, EG&G Bionomics
Dr. M. Lewis, Proctor and Gamble Co.
Dr. A. Maki, Exxon Corporation
Mr. B. McAllister, Analytical Bio-Chemistry Laboratories
Dr. C. Muska, E.I. Dupont DeNemours and Co.
Dr. A. Nebeker, EPA/ERL-Corvallis
Mr. M. Palmieri, Allied Chemical Corp.
Mr. B. Parkhurst, Western Aquatics, Inc.
Mr. J. Pearson, EPA/EMSL-Las Vegas
Dr. K. Porter, University of Georgia
Dr. W. van der Schalie, USAMBRDL, Ft. Detrick
Dr. C. Weber, EPA/EMSL-Cincinnati
Dr. L. Williams, EPA/EMSL-Las Vegas
In addition, thanks to the staff from Life Systems, Inc., Mr. G. Schiefer,
Dr. J. Glennon, and Ms. C. Patrick, for arranging and facilitating panel
meetings, transcribing proceedings, and documenting all panel activities;
and thanks to A. Pilli and S. Lozano, Montana State University, and R. Russo,
EPA/ERL-Duluth, for the initial working draft from which interim procedures
were developed. We also sincerely appreciate and thank Ms. T. Highland and
Ms. S. Heintz, Word Processing Center at Duluth and the Word Processing
Center, Computer Sciences Corporation, Las Vegas, for providing the many
typed iterations of these procedures.
viii
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SUMMARY
Adult daphnids in cultures used for providing young for testing must be
healthy and free of ephippia. Culture mortality of adult organisms must not
exceed 10 percent during the 14 days prior to testing. Culturing and testing
are conducted at a constant temperature of 20 _+ 1°C with a 16 hour photoperiod.
Daphnids are cultured and tested in hard reconstituted water for dilution
(American Public Health Association et al., 1980) and fed trout food and
Selenastrum capricornutum.
A 48-hour screening test may be used as a range-finder prior to an acute
test for samples in limited quantity or if nothing is known about the toxicity.
The screening test is conducted using a control and 1, 10, and 100 percent of
the leachate or effluent being tested (or a wide range of concentrations when
testing other forms of toxicants). Five <24-hour-old Daphm'a magna; in 80 ml of
solution are used in each 100 ml beaker at a test concentration. A 48-hour
static acute test is also conducted using five <24-hour-old Daphm'a per 80 ml of
solution in a 100 ml beaker. Five or more concentrations and a cont\rol (plus
an acetate or alternative solvent control, if needed) are tested in Quadrupli-
cate (i.e., there are 20 daphnids per experimental condition). The daphnids
are tested unfed. Immobilization or death is recorded at test termination, and
a 48-hour £650 concentration is calculated. The beakers in both tests are
covered with glass to minimize evaporation.
The 21-day chronic test is conducted using ten 100-mL beakers, each of
which contains one <24-hour-old daphnid in 80 ml of solution, at each concen-
tration tested. The acute £650 values are used as the basis for selecting the
test concentrations to use in the chronic test. The solution is changed, and
endpoints are recorded three times weekly (Monday, Wednesday, and Friday).
Temperature is monitored continuously. Dissolved oxygen, pH, hardness, and
alkalinity are measured initially and on 2- or 3-day-old samples when the
solution is renewed. The daphnids are fed 5 mg/L of trout food which must be
obtained from the Environmental Research Laboratory in Duluth, Minnesota, plus
108 cells/L (105 cells/mL) of Selenastrum capricornutum three times weekly
(Monday, Wednesday, and FridayJTEndpoints determined for each test include
survival and the number of young produced by each female that is alive at the
end of the test period. An optional endpoint is the length of those adult
daphnids that are alive at the end of the experiment.
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CULTURE AND TESTING METHODS
Daphnia magna are recommended because of their sensitivity to toxic
substances, large size, ease of identification, availability from laboratories
and commercial services, ease of handling, and extensive use in toxicity test-
ing. Daphnids must come from an established laboratory culture. Daphnia
tested in any toxicant must not be retained for culturing or testing with other
toxicants.
The trout food for these tests must be obtained from the Environmental
Research Laboratory-Duluth (ERL-D) where it has been tested (see Appendix G-2).
GENERAL CULTURE PROCEDURE FOR BROOD STOCKS
Daphnia magna may be cultured in 2,000-mL glass containers, each having
20 daphnids per 1,600 mL of hard (160-180 mg/L CaCOs) reconstituted water
(American Public Health Association et al., 1980; also see Appendix A). The
culture must be maintained at 20 _+ 2°C in a constant temperature bath (or in
an incubator or room regulated to narrow temperature tolerances) and exposed
to a 16-hour photoperiod. The Daphnia must be transferred to fresh water
weekly (minimum) and must be fed 5 mg/L of trout food (Appendix B) plus 10^
cells/L (10^ cells/mL) of Selanastrum capricornutum (Appendices C and D) each
Monday, Wednesday, and Friday (this number of cells is equivalent to about
1.8 mg dry weight of Selanastrum). At the time of transfer, only adults are
transferred; the young are disposed of or used to initiate additional cultures.
The young from the second to sixth broods from these adults are used to start
new cultures each week. When the adults are 4 weeks old, they are disposed of.
If new cultures are initiated every 7 days there will be a continuous source of
animals ready for acclimation. Maintaining cultures by this method minimizes
overcrowding, male production, ephippia formation, and population "crashes."
It also helps to control bacteria and fungi.
For transferring Daphnia use pipettes that have an inside diameter at
least 1.5 times the size of the animals. Care must be taken not to bump
or bruise the daphnids while transferring them to new media; they must be
introduced below the surface of the new medium to avoid trapping air under
their carapaces.
ACCLIMATION CULTURE PROCEDURES
Organisms
Adult daphnids (brood stock) about to have their second to sixth broods
are cultured under conditions similar to those for chronic tests. The brood
stock must be healthy. Their health is indicated by: survival; absence of
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floaters; absence of ephippia; large size of adults; dark coloration; absence
of external parasites; and presence of large numbers of young (three or more
young per female per reproductive day). Young daphnids produced from these
adults are then transferred into new'media and reared for at least two weeks.
These animals must be healthy as indicated by the criteria given above. Young
from these daphnids are then used for both acute and chronic tests.
Food and Feeding
Animals must be fed 5 mg/L of trout food and 108 cells/L Selenastrum
capricornutum three times each week (i.e., when the medium is changed).
Methods
Offspring (<24 hours old) of the adults set aside for acclimation must be
placed in culture chambers and must be subjected to test conditions for at
least 14 days. Culture vessels for acclimation must provide 80 ml of water per
animal and must be covered (not sealed) to minimize evaporation. Daphnids must
be transferred into clean containers every Monday, Wednesday, and Friday, when
the medium is changed, by using a fire-polished pipette; all transferring must
be done under the water surface. Note the survival of the test animals each
time the medium is changed. Mortality must not exceed 10 percent if the animals
are to be used for producing young to start an experiment. Reproduction must
be noted by counting the number of young when the medium is changed. The young
used for starting an experiment must come from the second to the sixth broods.
Containers
One-hundred-mi Hi liter glass containers (beakers are usually used) that
contain 80 ml of the dilution water (medium).
Replication
A sufficient number of replicates to assure that enough healthy young
daphnids are available to begin a test.
Aeration
Must not be used.
Cleaning
All glassware must be scrubbed with a 1-percent solution of Liquinox or
another non-phosphate detergent, rinsed with tap water until sudsing has ceased,
and then rinsed three more times with tap water. The glassware is then rinsed
three times with distilled water, once with 10 percent HN03, once with distilled
water, once with acetone, and six more times with distilled water.
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Light and Photoperiod
Fluorescent light bulbs must provide a color rendering index >90 with a
16-hour photoperiod automatically controlled. Do not exceed a range of
light intensity from 30 to 100 foot candles. Close regulation in the range of
50 to 70 foot candles is recommended to stabilize growth rates of the live
alga Selenastrum capricornutum used as feed for the daphnids.
Temperature
An instantaneous temperature of 20 +_ 2°C must not be exceeded; the daily
mean temperatures must be 20 _+ 1°C. Temperature should be monitored continuously
or should be measured with a maximum-minimum thermometer.
Water Quality Measurements
Hardness, alkalinity, and pH measurements must be made on each batch of
water used.
£H
The pH must be between 6.8 and 8.5.
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ACUTE TESTS
SPECIFIC PROCEDURES
All data will be recorded by using the form provided in Appendix F (1 and 2),
Organisms
Young daphids used for testing must come from the second to sixth broods
of laboratory-reared animals from healthy cultures.
Food and Feeding
Do not feed for acute tests.
Methods
Place young (<24-hour-old) Dapnnia in the chambers and subject them to
test conditions for 48 hours. Dapnnia must be transferred with a fire-polished
pipette (with an inside diameter about 1.5 times the size of the daphnids) into
beakers which then must be covered with a pane of glass or a watch glass to
minimize evaporation.
Containers
Use 100-mL borosilicate glass beakers containing 80 ml of test solution.
Leachates
Leachates (effluents or toxicants) must be stored at 4°C in the dark but
must be allowed to come gradually to 20 _+ 1°C before adding daphnids. Dilutions
are made in volumetric flasks and are then poured into the test beakers.
Dilution Water
Dilution water must be the same as the acclimation and culture water.
Controls
Controls must be set up and treated the same as test solutions, with regard
to experimental conditions, except that no leachate (toxicant, etc.) is added.
No more than 10 percent mortality may occur in 48 hours among control daphnids
for the test to be valid.
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Acetate Controls
Acetate controls must be run in addition to water controls whenever acetate
is used in generating the solid waste leachate to be tested. The acetate concen-
tration in the control and all concentrations tested must be the same as that
in the highest concentration. No more than 10 percent mortality may occur in
48 hours among acetate-control daphnids for the test to be valid. (If other
solvents are used, the same procedure is applicable.)
Test Concentrations
At least five toxicant concentrations with a dilution factor of 0.5
(e.g., 100 percent, 50 percent, etc.) or greater (0.75, e.g., 100 percent,
75 percent, 56 percent, etc.) must be used for 48-hour tests. The highest
concentration to test may be determined by a 48-hour screening test using
order of magnitude leachate (effluent) dilutions (i.e., 100 percent, 10 percent,
and 1 percent), with five daphnids in 80 ml of solution for each concentration
and control. The screening test solutions do not need to be duplicated but
will aid in determining the appropriate concentrations to use in the 48-hour
acute test. For example, if all animals die at 100 percent of the leachate and
if no animals die at 10 percent, then the following concentrations should be
tested for 48-hour acute tests: 100 percent, 50 percent, 25 percent, 12.5
percent, and 6.25 percent. (See Standard Methods [APHA, 1980] for suitable
toxicant concentrations.)
Randomization
Daphnids are transferred randomly from the acclimation stock to beakers
containing the appropriate experimental conditions. The beakers are, in turn,
placed at random locations in a water bath or a controlled temperature incubator
or room.
Replication
Four containers, each containing five daphnids (for a total of 20 animals),
are required for each experimental condition.
Aeration
Must not be used.
Cleaning
All glassware must be thoroughly washed with a laboratory detergent and
must be rinsed with tap water. Because most leachates are unknown mixtures,
a 10 percent nitric acid rinse which is followed by both a distilled water and
an acetone rinse and then is followed in turn by at least three distilled water
rinses is required. Test containers and flasks should have an additional rinse
with the dilution water to be used for testing just before a test is started.
(If testing only inorganic toxicants, acetone is not needed; if testing only
organic toxicants, nitric acid is not needed.)
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Light and Photoperiod
Fluorescent light bulbs must provide a color rendering index ^90. A light
intensity of 30 to 100 foot candles must be used with a controlled 16-hour
photoperiod.
Temperature
An instantaneous temperature of 20 _+ 2°C must not be exceeded; the daily
mean temperature must be 20 _+ 1°C. Temperature must be monitored continuously.
Water Quality Measurements
Hardness and alkalinity must be measured in the high and low concentrations
and in the control at 0-hour. Dissolved oxygen and pH measurements must be
made at 0 and 48 hours in the high, middle, and low concentrations, and the
control. Control concentrations for hardness, alkalinity, and pH of the hard
reconstituted water should be as follows: 170 _+ 10 mg/L CaC03, 115 +_ mg/L
CaC03; and 7.6-8.5, respectively (American Public Health Association et al.,
1980); dissolved oxygen must be from 90 to 100 percent saturation at the time
the test is started.
£H
The pH of the test water must be from 7.6 to 8.5. If the pH of the leachate
(toxicant, etc.) is initially between 6.8 and 8.5, no adjustments are required.
If not, the pH of each concentration tested must be adjusted by using sodium
hydroxide to raise the pH to j>6.8 or by using hydrochloric acid to lower the pH
to £8.5. The pH must be measured and, if needed, adjusted just prior to
beginning the acute test.
Leachate Measurements
Test solutions of leachates or effluents should be measured either
directly or indirectly. If leachates have had preliminary chemical analyses,
one of the dominant constituents (e.g., ammonia) may be measured to check
dilution; if not, either conductivity or total organic carbon may be used.
Concentrations of known individual toxicants must be measured directly.
Test Apparatus
Test equipment should consist primarily of high grade borosilicate glass
or stainless steel. Fluorocarbon and high-density polyethylene equipment is
acceptable if the toxicant tested is an inorganic chemical or mixture. Rubber
and plasticized materials must be avoided.
GENERAL ACUTE TEST PROCEDURE
1. Transfer parent generation to new culture beakers containing food
24 hours prior to the start of the test to ensure that only <24-hour-
old daphnids will be available for testing.
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2. Prepare test solutions by adjusting the temperature to 20 _+ 1°C and
by adjusting the pH to 6.8 to 8.5, if needed.
3. Label all test beakers.
4. Prepare test solutions by making the appropriate dilutions.
5. Fill test beakers with appropriate test solutions. The test commences
when the first animal is added; therefore, the time must be recorded.
6. Randomly add <24-hour-old daphnids into each beaker until each beaker
contains five daphnids. This should be accomplished in less than one
hour.
7. Randomly place control and test concentrations into rows; then
randomly place the beakers within each row and place a glass cover on
each.
8. At the end of 24 and 48 hours, count and record the number of
immobilized or dead Daphm'a per beaker.
9. Measure dissolved oxygen, pH, hardness, and alkalinity of the control
and of the highest concentration, and of intermediate concentrations
if the highest concentration is different from the control, at the
beginning and at the end of the test.
10. Measure test concentrations either directly or indirectly at the
beginning and at the end of the test.
11. Calculate the 48-hour ECsg and its 95 percent confidence limits
unless 100 percent of the leachate or effluent is nontoxic. If when
testing individual toxicants the highest concentration is not toxic,
repeat the test unless the solubility in water is exceeded.
STATISTICAL EVALUATIONS
An acceptable ECsg test will have at least two test concentrations where
the number of immobile (dead) animals bracket 50 percent unless there is less
than 50 percent response in the 100 percent solution or at the solubility limit
of the toxicant in water. If the lowest test concentration results in excess
of 50 percent response, the test must be repeated.
The analysis of the data must include:
1. A preliminary scatterplot of the response rates (number of dead or
immobile/48 hour) observed in each test or control beaker, versus
group number and percent of logarithm of concentration, to look for
patterns of response and outlying beakers.
2. ECso estimates based on the responses in the treatment groups, unless
they cannot be calculated for the reasons stated previously. EC$Q
estimates should be accompanied by estimates of their standard errors
8
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and of their 95 percent confidence intervals. In the event that the
confidence intervals are very wide (e.g., if the concentration effect
curve is very shallow), the highest concentration for the chronic
test should be chosen below the
3. The results of outlier tests, as described below, and the preliminary
scatterplot should be used to detect outlying beakers (clearly atypical)
within a treatment or control group.
4. If the results from one or more beakers are determined to be outliers,
then £650 estimates, standard errors, and confidence intervals will
be calculated both by including and by excluding these values.
The experimental records corresponding to suspected outliers will be
examined. If these records are found to contain clerical or experimental
errors leading to erroneous values, the erroneous values will be corrected or
will be discarded as appropriate, and the analyses will proceed. If the
outlying values are not the obvious result of such errors, an outlier detection
test (Miller, 1966, Barnett and Lewis, 1978) will be carried out. If the
outlier test declares the value to be an outlier, then subsequent analyses will
be carried out both with and without that value, and both sets of estimates
will be reported. If the outlier test does not declare the value to be an
outlier, then all subsequent analyses will include the suspect value.
Acceptable methods of estimating the EC§Q include the two-parameter probit
or logit methods (Finney, 1978) and the trimmed Spearman-Karber method (trimming
proportion must be reported, Hamilton et al . , 1977). The method of estimation
used must be specified along with any assumptions or discretionary adjustments
that are used. Use of any other method of estimation must be justified by
citing generally accepted references in which the estimation method is described
and recommended for similar testing situations.
CHRONIC STATIC-RENEWAL TESTS
Specific Procedures
All data wil be recorded by using the form in Appendix F (3 and 4).
Organisms —
Test animals must come from a healthy culture and must be raised under
controlled acclimation conditions for a minimum of 14 days prior to the start
of a test. Parental organisms about to have their second to sixth broods must
be transferred into new media less than 24 hours prior to starting a test.
Food and Feeding--
Trout food (5 mg/L) plus Selenastrum capricornutum (108 cells/L) are
required. Food must be added with the toxicant in the flask initially and when
test solutions are renewed (three times each week).
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Methods--
Young (<24-hour-old) daphnids must be placed in test chambers and sub-
jected to test conditions for 21 days. Ten 100-mL beakers are used for each
experimental group for each test. One daphm'd is placed in each beaker con-
taining 80 mL of test solution. The beakers must be distributed randomly. The
beakers must be covered with a glass cover (plate or watchglass) to minimize
evaporation and to keep out debris. Daphnids must be transferred into clean
containers every Monday, Wednesday, and Friday when the medium is changed by
using a fire-polished pipette (with the inside diameter about 1.5 times the
size of the animal being transferred); all transferring of organisms must be
done under the surface of the water. Survival of the test organisms must be
noted each time the medium is changed. Reproduction must be noted by counting
the number of young; the young must be counted and discarded each time the
adults are transferred and at the end of each experiment.
Containers—
100-mL borosilicate glass beakers containing 80 ml of control or test
solution.
Leachate—
Leachate (or effluents to be tested) must be stored at 4°C in the dark but
must be allowed to come to 20 +_ 1°C before adding daphnids. Dilution of the
leachate or toxicant solution and the mixing of food are best accomplished in
volumetric flasks; the solutions can then be poured into test containers. The
solutions must be renewed three times each week; this is best accomplished by
adding food and toxicant (test) solution to clean beakers and then transferring
adult daphnids to the resulting test cells. Daphnids must be added within one
hour after the test solutions have been prepared.
Dilution Water—
Dilution water must be the same as that used for culturing.
Controls--
Controls must be set up and treated the same as test containers with
regard to experimental conditions except that no leachate (toxicant, etc.) is
added. Control animals must produce a minimum average of 40 young in 21 days
for the experiment to be valid. At least 80 percent of the adults must survive
in the control water for the 3-week test period for the test to be valid.
Acetate Controls--
Acetate controls must be run in addition to water controls whenever acetate
is used in generating solid waste leachate for testing. The acetate concentration
in the control and all concentrations tested must be the same as that in the
highest leachate concentration. At least 80 percent of the adults must survive
for 21 days in the acetate control solution for the test to be valid. If other
solvents are used, the same procedure (solvent control testing) is applicable.
Concentrations—
The test design should include at least five concentrations of test
material (toxicant) made up in a geometric progression with a dilution
factor of 0.5 (e.g., 100 percent, 50 percent, 25 percent, etc.) or greater
(100 percent, 75 percent, 56.25 percent, etc.). Initial concentrations selected
10
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for testing should bracket (i.e., above and below) previous results or should
be based on results from acute tests (let the highest test concentration equal
the 48-hour
Randomization —
Daphnids are randomly assigned from the acclimation stock to the test beakers.
A two-stage transfer procedure is needed. Daphnids from the culture stock are
randomly transferred into beakers containing dilution water. A second transfer
is then made into beakers containing the appropriate experimental conditions.
The order of assignment is determined from a table of random numbers or from
another method of random allocation. The control and test concentrations are
then randomized into rows, and the beakers are randomized within each row.
Replication--
Ten containers, each containing one daphnid (a total of ten animals), are
required for each experimental condition.
Aeration--
Must not be used.
Cleaning —
All glassware must be cleaned as follows: scrub with a 1 percent solution
of Liquinox or other suitable non-phosphate detergent, rinse with tap water
until sudsing has ceased, then rinse three more times with tap water. Rinse
three times with distilled water, once with 10 percent HN03, once again with
distilled water, once with acetone, and finally six more times with distilled
water.
Light and Photoperiod--
Fluorescent light bulbs must provide a color rendering index >90 with a
closely controlled 16-hour photoperiod. A light intensity of 30 to 100 foot
candles must be used. The narrower range of 50 to 70 foot candles may be
desirable because green algae acclimated to low light conditions can photo-
sythesize at a rapid enough rate toward the upper end of the allowable
range that pH levels increase to or beyond the recommended pH range. If
that occurs, raise or mask the light source to reduce effective illumination
until the pH range stabilizes within the recommended limits.
Temperature —
An instantaneous temperature of 20 _+ 2°C must not be exceeded; the
daily mean temperature must be 20 _+ 1°C. The temperature must be monitored
continuously.
Water Quality Measurements —
Hardness, alkalinity, pH, and dissolved oxygen measurements must be made
for the high and low concentrations and the control when solutions are prepared.
Dissolved oxygen and pH measurements must be made in the high, middle, and low
concentrations after transfer of daphnids on 2-3-day-old solutions. In addition
to the above measurements, the dissolved oxygen must be measured the morning
after solutions have been added before the lights come on; this should be
accomplished by setting up an additional control (i.e., set up additional
11
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controls once or twice during the experiment specifically for checking dissolved
oxygen).
PH«
The pH of the leachate (or toxicant, etc.) to be used in testing must be
adjusted (so that the resulting test solution is within the recommended range)
by using sodium hydroxide to raise the pH to 6.8 or by using hydrochloric acid
to lower the pH to 8.5. The pH of test solutions must be measured and adjusted
prior to the beginning and just before each renewal for chronic tests. If the
pH is initially between 6.8 and 8.5, no adjustments are required.
Leachate (Effluent or Toxicant Solution)—
Test solution concentrations should be measured either directly or
indirectly. If leachates or effluents have had preliminary chemical analyses,
one of the dominant constituents (e.g., ammonia) may be measured to check
dilutions; if not, either conductivity or total organic carbon may be used.
Individual toxicant concentrations must be measured directly.
Test Apparatus—
Test equipment should consist primarily of high grade borosilicate glass
or stainless steel. Fluorocarbon and high density polyethylene equipment are
acceptable if the chemical or mixture being tested is inorganic in nature.
Equipment constructed with rubber or plasticized materials that could contact
tests solution must be avoided.
General Chronic Test Procedures
1. Transfer parent generation to new culture beakers containing food 24
hours prior to the start of a test to ensure that only <24-hour-old
young will be available for testing.
2. Prepare dilutions in volumetric flasks and add dilution water nearly
up to the desired volume.
3. Add trout food plus Selenastrum to volumetric flasks, make up to the
appropriate volume (usually 1 liter) with reconstituted water, and
mix well.
4. Carefully label all beakers.
5. Fill test beakers with 80 ml of the appropriate test solutions.
6. Randomly add <24-hour-old daphnids into each beaker until all beakers
contain one Daphnia and note the time when the first daphnid is added.
7. Randomly place control and test concentration beakers into rows, and
place beakers in random positions within each row; then cover with
glass, and record the time.
12
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8. Every Monday, Wednesday, and Friday:
a. Count number of dead or immobilized adults.
b. Mix fresh test solutions containing food for each
experimental condition.
c. Pour test solutions into clean beakers and transfer
daphnids.
d. Count number of young per surviving female.
e. Discard dead adults and all young.
9. Measure dissolved oxygen, pH, hardness, and alkalinity when the
experiment is set up and measure dissolved oxygen and pH on 2- or
3-day-old samples. Perform sufficient (a minimum of six times during
an experiment) measurements on subsequent set-ups to characterize
these parameters.
10. Record and evaluate adult mortality and young per female for animals
living 21 days by using the appropriate statistical procedures.
As an option, measure the length of Daphnia surviving at the end of
the experiment.
STATISTICAL EVALUATIONS
Statistical analysis of the chronic test results will be carried out for
the mortality and reproduction responses. Statistical analyses of body lengths
may be presented at the discretion of the investigator. Analyses of reproduction
and growth (length) responses will be carried out only on those daphnids that
survive to the end of the test.
For analysis of the mortality results, a distinction will be made between
toxicant-related and accidental mortality. The causes, if known, of all accident-
related deaths will be documented. Accident-related deaths per treatment level
must not be >20 percent of the daphnids tested. Final (21-day) mortality
results will be adjusted for accident-related mortality by disregarding those
daphnids that died (i.e., those daphnids are excluded from both the numerator
and the denominator when calculating the toxicant-related mortality rates in
each group).
Results of the statistical analyses on the mortality, reproduction, and
length responses will be presented in terms of a no-observed effect concentra-
tion (NOEC) and of a statistically significant effect concentration. The NOEC
concentration is the highest test concentration at and below which the average
response does not differ significantly from the control group response. The
statistically significant effect concentration is the next highest concentration.
Estimates of the LCt>Q for toxicant-related mortality, along with associated
standard errors and confidence intervals, will be reported.
13
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Survival
Preliminary scatterplots will be prepared of the toxicant-related mortality
rates versus group number, concentration, or the logarithm of concentration.
The proportion of toxicant-related deaths within each group will be
calculated by dividing the number of toxicant-related deaths at 21 days by
group size minus the number of accidental deaths. Each such proportion, p,
will be transformed by the arcsine variance-stabilizing transformation to
[arcsin /r/n+1 + arcsin /(r+l)/(n+l)] for small sample sizes. The transformed
proportions will be tested for equality by a one-way analysis of variance.
Multiple comparisons between each treatment group and the solvent or acetate
control group will be carried out by Dunnett's many-one t procedure or by the
Bonferroni t procedure (Miller, 1966) to determine which treatment groups have
significantly different mortality rates (at the 95 percent confidence level)
from the control group.
The LCso and LCjo concentrations and associated standard errors and
confidence intervals may be estimated by any of the methods discussed for the
acute test. The trimmed Spearman-Karber method is appropriate for estimating
the LC50.
Reproduction and Length
The statistical analyses of reproduction and length will be similar to one
another. Analyses will be confined to 21-day survivors. Reproduction will be
reported as the total number of offspring per female for animals living 21 days.
Lengths will refer to 21-day lengths.
Preliminary scatterplots of individual responses versus group number, con-
centration, or the logarithm of concentration will be prepared. Group average
responses will be included in these displays. These plots will be examined to
determine the nature of the relation between concentration and average response,
the relation between average response and standard deviation, and the presence
of outliers.
The experimental records will be examined for suspected outliers. If
these records are found to contain clerical or experimental errors leading to
erroneous values, the errroneous values will be corrected or discarded and the
analysis will proceed. If the outlying values are not the obvious result of
such errors, an outlier detection test (Miller, 1966, Barnett and Lewis, 1978)
will be carried out. If the outlier test declares the value to be an outlier,
then susequent analyses will be carried out both with and without the response,
and both sets of estimates will be reported. If the outlier test does not
declare the value to be an outlier, then all subsequent analyses will include
the suspect value.
If the variability appears to vary from group to group in the preliminary
scatterplots, use a log transformation. The original or transformed average
values within each group will be tested for equality by a parametric or
nonparametric one-way analysis of variance.
14
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Parametric or nonparametric multiple comparisons between each treatment
group and the solvent or acetate control group will be carried out by Dunnett's
many-one t procedure or by the Bonferroni t procedure (Miller, 1966) or by the
Kruskal-Wallis rank-sum-based procedure (Hollander and Wolfe, 1973) to deter-
mine which treatment groups have significantly different response rates (at the
95 percent level) from the control group.
CONFIDENCE INTERVALS AND AFTER-THE-FACT POWER CALCULATIONS
The determination of NOECs and statistically significant concentrations
does not impart information about the sensitivity of the inferences, i.e., an
insensitive test might not reveal statistically significant differences in
group average responses even when the differences are clearly of biological
significance.
After-the-fact power calculations can be carried out to determine how
large a treatment response must be before it can be statistically differentiated
from the control response with high probability. Power calculations for length
and productivity responses will be based on the noncentral t distribution when
adjustment is made for multiple comparisons by Bonferroni's method. Power
calculations for mortality responses will be based on Fisher's exact test
(Bennett and Hsu, 1960; Haseman, 1978).
Confidence intervals (95 percent) on the differences between the average
responses in the solvent or acetate control group and those at the NOEC or
statistically significant concentration will be prepared. Confidence inter-
vals for the reproduction and length responses which account for multiple
comparisons and for possibly heterogeneous variances will be based on the t-
distribution. Confidence intervals for mortality responses will be based on
the Poisson approach (Feder, 1981; Nelson, 1970) and will account for multiple
comparisons.
15
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OBTAINING AND RECORDING DATA
ACUTE
After 24 hours and at the completion of the acute test, the number of dead
and immobile daphnids in each beaker must be counted so that an ECso can be
determined. If calculating an optional \-C$Q, the daphnids that are immobile
must be carefully transferred with a glass pipette into a petri dish or watch
glass. Using a SOX dissecting microscope, observe each daphnid individually
for heartbeat. Absence of a heartbeat will denote a dead daphnid and will
provide data for the determination of an LCsg.
CHRONIC
The number of dead adult Daphnia are counted by observation only (no
microscopic examination required).
The number of young are most easily counted by removing them with a pipette
from the test beaker after the adult has been transferred and by counting them.
An automatic counter is not recommended as this will count food particles, etc.,
which are similar in size to the daphnids.
If length measurements are to be used, adult daphnids alive at the end
of the test are measured using a SOX compound microscope with a calibrated
micrometer eyepiece insert.
A 21-day LCsg and the number of young per female for animals surviving 21
days must be reported. The lowest concentration causing an effect on reproduc
tion at the 95 percent confidence level must be reported and will constitute
the toxic concentration. The next lower concentration will constitute the
no-significant-effect (NOEC) concentration at the 95 percent confidence level.
16
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DATA REPORTING
(Adapted from Peltier 1978)
A report of the test results must include:
o The name of the test method, investigator, and laboratory.
o A description of the leachate effluent or toxicant, including
its source and any physical and chemical properties known.
o A description of the extraction procedure used if testing a
leachate.
o The chemical characteristics of the dilution water.
o The scientific name and the source of the test organism.
° A description of the test procedure.
o The methods used for measuring hardness, alkalinity, dissolved
oxygen, pH, and temperature, and the results of these measurements,
o Direct or indirect measurements of leachates, effluents, or
toxicants.
o Methods used for all chemical analyses.
For acute test results:
o A description of the endpoint used and of the results of the
statistical analyses conducted.
o The percent of organisms that survived in each experimental
solution.
o An ECso value and the 95 percent confidence limits.
o The methods used for statistical analyses of the data.
For chronic test results:
o The number of mortalities and effects observed in controls.
17
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A nonsignificant and a significant effect concentration at the
95 percent confidence level for the number of young produced by
each female that survived 21 days.
A 21-day LCsg with 95 percent confidence limits.
Methods used for statistical analyses.
Behavioral or other relevant information.
18
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LITERATURE CITED
American Public Health Association, American Water Works Association, Water
Pollution Control Federation. 1980. Standard methods for the examination
of water and wastewater. 15th ed. New York, N.Y. 1134 p.
Barnett, V.t and T. Lewis. 1978. Outliers in statistical data. John Wiley
and Sons, New York, N.Y.
Bennett, B. M., and P. Hsu. 1960. On the power function of the exact test for
the 2X2 contingency table. Biometrika 47: 393-398.
Brown, M. B., and A. B. Forsythe. 1974. Robust tests for the equality of
variances. J. Am. Stat. Assoc. 69: 364-367.
Feder, P. 1981. Design and analysis of chronic aquatic tests of toxicity with
Daphnia magna. The report of the Battelle Columbus Laboratory to the U.S.
Army Medical Research and Development Command. 431 p.
Finney, D. J. 1971. Probit analysis, third edition. Cambridge University
Press, London.
Finney, D. J. 1978. Statistical method in biological assay. Charles Griffin
and Company, London, England.
Hamilton, M. A., R. C. Russo, and R. V. Thurston. 1977. Trimmed Spearman-Karber
Method for estimating median lethal concentrations in toxicity bioassays.
Environ. Sci. Technol. 11: 714-719. Correction: Ibid. 12: 417 (1978).
Haseman, J. K. 1978. Exact sample sizes for use with the Fisher-Irwin test
for 2X2 tables. Biometrics 34: 106-109.
Hollander, M., and D. A. Wolfe. 1973. Non-parametric statistical methods.
John Wiley and Sons, New York, N.Y.
Miller, R. G. 1966. Simultaneous statistical inference. McGraw-Hill Book
Company, New York, N.Y.
Nelson, W. 1970. Confidence intervals for the ration of two Poisson means
and Poisson prediction intervals. I.E.E.E. Transactions on Reliability.
R-19: 42-49.
Neter, J., and W. Wasserman. 1974. Applied linear statistical models.
Richard D. Irwin, Inc., Homewood, Illinois.
19
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Peltier, W. 1978. Methods for measuring the acute toxicity of effluents to
aquatic organisms. Environ. Monit. Series, EPA-600/4-78-012, Environ.
Monit. Support Lab., U.S. Environ. Prot. Agency, Cincinnati, Ohio: 51 p.
Williams, D. A. 1972. The comparison of several dose levels with a zero dose
control. Biometrics 28: 519-531.
Williams, L. R., K. Biesinger, W. van der Schalie, R. E. Bently, and
T. C. Surprenant. 1986. Collaborative study of Dapnia magna static
renewal assays. EPA/600/X-86/115. Environmental Monitoring Systems
Laboratory, Las Vegas, Nevada. 26 p. and App. A through H.
20
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REFERENCES
Biesinger, K. E. 1975. Tentative procedure for Daphnia magna chronic tests
in a standing system. Federal Register 40 (123): 26902-26903.
Biesinger, K. E., and G. M. Christensen. 1972. Effects of various metals on
survival, growth, and reproduction of Daphnia magna. J. Fish. Res. Board
Can. 29(12): 1691-1700.
Epler, J. L., et al., 1980. Toxicity of leachates. Oak Ridge National
Laboratory, Oak Ridge, TN, EPA-600/2-800-057, U.S. NTIS PB-80-179328:
133 p.
Goulden, C. E., R. M. Comotto, J. A. Hendrickson, Jr., L. L. Hornig, and
K. L. Johnson. 1982. Procedures and recommendations for the culture
and use of Daphnia in bioassay studies. Special Technical Publication
No. 766, Aquatic Toxicology: 5th Conference ASTM. pp 139-160.
Murphy, J. S. 1976. A general method for the monoxenic cultivation of the
Daphnidae. Biol. Bull. 139: 321-332.
Nebeker, A. V. 1982. Evaluation of the Daphnia magna renewal life-cycle test
with silver and endosulfan. Water Res. 16: 739-744.
Organization for Economic Co-operation and Development. 1981. Guidelines for
testing of chemicals: Daphnia sp., 14-day reproduction test (including
an acute immobilization test). Organization for Economic Co-operation and
Development, Paris, France.
U.S. Environmental Protection Agency. 1982. Occupational health and safety
manual, Chap. 8: Laboratory use-of toxic substances. Office of
Resource Planning and Management, U.S. Environmental Protection Agency,
Washington, D.C.
21
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APPENDIX A
RECONSTITUTED HARD WATER PREPARATION*
Materials Needed:
1. 5 gallon carboy
2. deionized distilled water
3. chemicals
o NaHCOa
o CaS04.2H20
o KC1
4. weighing pans and spatula
5. balance (accurate to 0.001 gram)
6. storage jars for salts (optional)
Methods:
1. Thoroughly rinse the 5 gallon carboy with a 10 percent solution of
nitric acid. Slowly pour out acid solution into cold running water.
Rinse carboy thoroughly with deionized distilled water at least five
times. Accurately mark the 19 liter level in the carboy to facili-
tate preparation of water each time.
2. Weigh out stock chemicals one at a time in the following amounts:
3.65 g NaHC03
2.28 g CaS04.2H20
*The 15th edition of Standard Methods (American Public Health Association
et al., 1980, p. 627) has a table for hard reconstituted water.
22
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2.28 g MgS04
0.15 g KC1
Extra stock mixtures can be weighed out Tn advance for use in the
next week if stored in tightly covered jars.
3. Add approximately 15 liters of deionized distilled water to the
carboy. Add the chemicals in the order given, and mix thoroughly
after each addition. Rinse storage jar with deionized distilled
water and add rinse water to solution in carboy. Mix solution
thoroughly. Add deionized distilled water to a total solution
volume of 19 liters.
4. To assure complete mixing of chemicals and saturation with dissolved
oxygen, stir with the lid removed (but covered with a foam plug or
glass wool) for 24 hours using a magnetic stirrer.
5. Measure hardness, alkalinity, dissolved oxygen, and pH. The hardness
must be from 160-180 mg/1 CaC03; the alkalinity from 110-120 mg/1
CaC03; and the pH from 7.6-8.5. This will verify proper measurement
and mixing of salts in preparing the reconstituted water. If the
hardness, alkalinity, and pH requirements are not met, the reconsti-
tuted water must be prepared again.
6. Reconstituted water may be stored and used for one month.
23
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APPENDIX B
DAPHNIA TROUT FOOD PREPARATION
- Add 15 grams of trout food (obtained from the Environmental Research
Laboratory-Diluth) to 800 ml of reconstituted hard water and blend for 15
minutes to liquify.
- Pour into a suitable container and add 200 ml of reconstituted hard water.
- Let stand for 15 minutes and then carefully decant the upper 800 mL and
discard the remaining precipitate.
- Thoroughly mix the suspension and withdraw three 10-mL aliquots.
- Dry the aliquots at 104°C for 24 hours in preweighed tares.
- Weigh dry samples and subtract tare weight.
- Calculate average weight of a dry sample and the standard deviation.
- Calculate weight for 1 mL of dry solids. The final concentration must be
5 mg dry solids per mL of food, and the volume must be adjusted by adding
water. The total volume of water (X) to add equals the number of mL in the
sample after removal of the aliquots (770 mL) times the mg/mL of dry food
weighed (Y) divided by the mg/mL of dry food desired (5 mg/mL) minus the
number of mL in the sample after the removal of the aliquots.
For example, if the dry food weighed 6.32 mg/mL (Y), the following
equation will give X:
(770) (y)
X = - 770 where Y = mg/mL dry weight
5
(770) (6.32)
X = 770
X = 203 mL of water to add to 770 mL to give a concentration of 5
mg/mL of dry food.
- Store prepared Daphnia trout food in refrigerator for up to one week.
24
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APPENDIX C
CULTURING SELENASTRUM CAPRICORNUTUM
Algae origin:
American Type Culture Collection
12301 Park!awn Drive OR
Rockville, MD 20852
The Starr Collection
Department of Biology
University of Texas at Austin
Austin, TX 78712
Algae type:
1. Selenastrum capricornutum ATC #22662
2. Selenastrum capricornutum UTEX1648
Maintenance conditions:
1. Constant temperature from 18 _+ 10°C to 24 _+ 10°C
2. Lighting continuous "cool-white" fluorescent light from 4000 +_ 10
percent to 5000 +_ 10 percent lumens; photoperiod from 14L:10D to
continuous lighting.
3. The cultures must be maintained sterile in a chemostat (flow-through)
system or have continuous aeration and must be stirred with a magnetic
stirrer or shaken on a suitable shaker.
Glassware Cleaning - All glassware used for any aspect of algal culturing
must be cleaned as follows: scrub with a 1 percent solution of Liquinox or
other non-phosphate detergent, rinse with tap water until sudsing has ceased,
then rinse three more times with tap water. Rinse three times with distilled
water, rinse once with 10 percent HN03, rinse with distilled water, rinse once
with acetone, and rinse six times with distilled water. Autoclave all glass-
ware to be used for all phases of algae culture.
25
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Synthetic algal media stock preparation Oa*
1. Macronutrient stocks. Prepare separate stocks (for Woods Hole MBL
medium) of each of the following compounds by dissolving the specified
weight into a total volume of one liter of glass-distilled water.
Compound Grams/Liter
CaCl2
CaC1110.2H20 36.76
MgS04.7H20 36.97
NaHC03 12.60
KI2HP04 8.71
NaN03 85.01
Na2Si03.9H20 28.42**
2. Micronutrient stocks. Prepare each stock solution shown below in
a final volume of one liter of glass-distilled water. Mix until
dissolved. For stock No. 3, add chemicals in the order shown.
Stock No. Compound Grams/Liter
1 Na2EDTA 4.36t
2 FeCl3.6H20 1.575tt
3 CuS04.5H20 0.01
CoCl2.6H20 0.01
ZnS04.7H20 0.022
MnCl2.4H20 0.18
Na2Mo04.2H20 0.006
H3B03 1.0
*0a The method is based largely on conversations with Dr. Clyde Goulden and
Ms. Linda Henry (Academy of Natural Sciences, Philadelphia) for Selenastrum
culture in micronutrient supplemented MBL medium.
**Filter sterilize this stock solution and add 1 mL to the culture medium
after autoclaving by making use of a sterile technique.
tStock must be less than 3 months old.
ttUse 2 ml/I of medium.
26
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3. Record stock solution preparation information. All compounds used
must be ACS Reagent grade (or other high purity grade if no ACS
standard has been established for the compound used). Refrigerate
all stocks. Stocks, other than sodium silicate, showing any evidence
of precipitation or contamination must not be used. Precipitation of
the sodium silicate may occur with time, but the stock can still be
used.
4. For each liter of culture medium being prepared, add one milliliter
of each macronutrient stock (except sodium silicate) and one milli-
liter (2 mL of FeCl2) of each micronutrient stock to ~900 ml of
glass-distilled water. Add nutrients in the order given in 1 and 2
above, stir between each addition, and then make up to a liter with
glass-distilled water. Place one liter of medium in a 2-L Erlenmeyer
flask and cap with a foam plug (Gaymar IDENTI-PLUGS are recommended -
Miller et al., 1978) or with a cotton plug wrapped in cheesecloth.
Cover the top with aluminum foil. Autoclave at 1.1 kg/cm 02 (15 psi)
and 1210°C for 15 minutes. Allow to come to room temperature. Add 1
ml Na2Si03.9H20 stock by using a sterile technique.
5. For agar slants and petri plates, prepare medium as above but, in
addition, dissolve 1 percent (w/v) agar (DIFCO Bacto-Agar or equiva-
lent) prior to autoclaving. Place agar solution into test tube for
slants; tilt after removal from autoclave but before the agar has
jelled. For obtaining uncontaminated algal cultures, pour autoclaved
solution into sterile petri plates by using a sterile technique.
Obtaining Uncontaminated Algal Cultures - If stock algal cultures become
contaminated or if it is necessary to obtain new uncontaminated algal stocks,
use the procedure described below.
1. Using a sterile pipette, transfer one drop of algae in algal medium
to a sterile petri plate with the appropriate agar medium. Streak
and allow colonies to grow.
2. Select a presumptive clean single cell isolate from the plate and
transfer to a new plate. Streak again. Use the uncontaminated
single cell isolates from this plate to start new agar slants.
Initiating and Growing Algal Cultures
1. Obtain uncontaminated cells from isolates as described above. Pre-
pare new agar slants by transfer from uncontaminated agar slants.
Sufficient agar slants should be prepared such that one is available
every time a new algal inoculum must be prepared. Keep slants for
three to six months; but discard after use in one set of transfers.
2. Make a new set of slants (as required) from an available uncontami-
nated slant, then inoculate 100 mL of medium with algae from the
slant. Allow the algae to grow in the medium and use the inoculum
prior to the stationary phase of growth. This may be determined by
27
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visual examination of the color of the medium once sufficient
experience is gained with culturing. Otherwise, a sample must be
withdrawn with a sterile pipette and counted with a hemacytometer to
ensure that the cells are in log-phase growth (it is assumed that
baseline data is available on the growth curve of the alga so that
the cell concentration at the beginning of the stationary phase of
growth is known).
3. Static cultures are prepared by inoculating a vessel of MBL with a
batch culture. Each vessel should be covered with a cotton stopper
and should be continuously aerated and stirred with a magnetic stir-bar
or should be placed on a shaker table. If this system is used in an
on-going feeding program, new vessels must be inoculated on a careful
schedule to insure that adequate supplies of algae are available at
all times.
4. The semi-continuous culture system is prepared by hooking a 4 or 9
liter reservoir of the culture medium to a 4 liter aspirator bottle
with a silicone rubber siphon. The aspirator is first inoculated
with a batch culture of algae, and culture media is then siphoned
from the reservoir placed above the aspirator bottle. When the
culture is ready for harvesting, algae can be removed for use and can
be replaced with fresh media as needed. Semi-continuous cultures
should not be used for more than one month. A similar but more
complex system for semi-continuous culturing is described in Chapter
15 of Stein's (1973) Phycological Methods. Air lines should have a
cotton-filled trap to absorb oil or toxic liquids.
28
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APPENDIX D
PREPARATION OF ALGAE FOR FEEDING DAPHNIDS
METHOD 1
A drop of algae from a well-mixed culture of Selenastrum is used to fill
a hemacytometer counting cell. Enough sets (having 16 squares each) are
scanned so that between 100 and 200 algae cells are counted. A conversion
of the number of cells counted into the number of cells per mi Hi liter is made
by using the following formula:
(No. of cells counted) x (4 x 1006)
= No. of cells/ml
No. of squares counted
The number of mi Hi liters needed to get 1008 cells is determined by dividing
1008 cells by the number of cells per ml in the culture. The volume (ml) thus
determined is then measured out, placed in centrifuge tubes, and centrifuged at
2,200 RPM (700 x g) for 15 minutes. The algae media is then carefully poured
off, and ten milliliters of reconstituted water is added to resuspend the algae
(i.e., 10 ml will then contain approximately 108 Selenastrum cells). The
resuspended Selenastrum is then added to volumetric flasks containing approxi-
mately 950 mL of leachate (test solution), fish food, and reconstituted water.
The centrifuge tubes are rinsed twice to assure that all algae are removed, and
the rinse water is then added to the test solution. The test solution is then
made up to one liter; it is now ready for dispensing into the test chambers.
Algae suspended in centrifuge cells may be stored in the dark at 40°C for 10 to
12 days for subsequent feeding to daphnids.
METHOD 2
Check cell concentrations to confirm log-phase growth. Centrifuge the
algae at a speed and time sufficient to remove the algae from the water column
(700 x g for 15 minutes is suggested). Pour off the supernatant and leave
behind as little of the algal medium as possible. Resuspend the algae in a
small amount of the same solution used for culturing the daphnids about to be
fed. Remove a small portion from the algal solution and dilute as needed to
perform a hemacytometer count. Count at least 100 cells per field; determine
the original cell concentration per milliliter as follows:
Cells/mL = (cell count) (10,000)
(25/the number of double-lined fields counted)
(dilution factor)
29
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Dilute the algal solution with the appropriate daphnid culture medium so that
milliliter of the resulting dilution, when added to 800 ml of daphnid medium,
will create the appropriate food concentration. Confirm the final cell
concentration with a hemacytometer count. Harvested Selenastrum may be stored
in the refrigerator for 10 to 12 days after harvest.
METHOD 3
A particle counter may be used for counting algal cells.
NOTE: If the algae appears yellowish, brownish, clumps heavily on the sides
of the culture vessels, or does not appear in the microscope as intact
cells, something is wrong with either the algal stock or your culture
technique. Common problems include errors in media preparation or heavy
contamination with some other organisms such as bacteria. If the above
problems occur, the algae cultures should be replaced. If they persist,
the media preparations should be replaced, and new slants should be
ordered from the collections mentioned earlier.
REFERENCES
Stein, J. 1973. Handbook of Phycological Methods. Culture Methods and Growth
Measurements. Cambridge University Press, Cambridge, England.
30
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APPENDIX E
EQUIPMENT
Equipment
Pipettes (daphnids)
Pipettes (algae)
Suction bulbs
Culture beakers
(daphnids)
Test beakers
(daphnids)
Erlenmeyer flasks
(algae)
Foam plugs (algae)
Carboys
Fluorescent lights
(algae and daphnid
maintenance)
Light table
Light meter
Dissolved oxygen meter
pH meter
Compound microscope
Model Specifications*
5-mm and 8-mm
1-mL x 1/100 Polystyrene plugged
sterile disposable
Rubber, 1/2 ounce
2000-mL glass containers
100-mL Pyrex or Kimax
1000- and 2000-mL
Pyrex or Kimax
Nontoxic foam plug
35-45 mm
5 gallon plastic w/spigot
"Cool-white" for algae
"Grown-Lux" and "Vita-Life"
for daphnids
Model GB 11-17
30 watts "Glow Box"
Model No. 200
Model 0260
Oxygen Analyzer
0-14 pH units
+ 1/10 pH
*0r equivalent
31
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Appendix E, Equipment (continued)
Dissecting microscope
Equipment
Micrometer
Hemacytometer
Centrifuge
Membrane filter apparatus
Autoclave or pressure cooker
Drying oven
Dishwasher
Balance
15 x W.F., Cat. 147
Model Specifications
0.01 or 0.001 inches
at 4X
Model Pr-2 1000 x g force
Temperature capability
1000°C
L/A-7537 glassware washer
Accurate to 0.0001 gram
32
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APPENDIX F
STATIC ACUTE TOXICITY TEST
FORMS
33
-------
FORM
STATIC ACUTE TOXICITY TEST
TEST MATERIAL
Date
Time
Control
Temp
Data By
REP
CONC
DO
PH
HRD
ALK
DO
HRD
ALK
(continued)
34
-------
FORM
STATIC ACUTE TOXICITY TEST (Continued)
TEST MATERIAL
STOCK CONCENTRATION
PREPARED BY
TEST CONCENTRATIONS
mg/L ug/L
Amount of
Stock Added
Amount of Dilution
H20 Added
SOLVENT
DATE
PREPARED
HIGH > LOW
CONT.
S. CONT.
(continued)
35
-------
FORM
STATIC ACUTE TOXICITY TEST
TEST MATERIAL
DATE
TIME
DATA BY
REP.
CONC.
mg/l ug/L
Control
0-HOUR
A
OBSER.
B
OBSER.
C
OBSER.
0
OBSER.
24-HOUR
A
OBSER.
NO.
DEAD
B
OBSER.
NO.
DEAD
C
OBSER.
NO.
DEAD
0
OBSER.
NO.
DEAD
CUM
NO.
DEAD
48-HOUR
A
OBSER.
NO.
DEAD
B
OBSER.
NO.
DEAD
C
OBSER.
DEAD
0
OBSER.
NO.
DFAD
CUM
NO.
DEAD
oo
(continued)
-------
FORM
TEST MATERIAL
STATIC ACUTE TOXICITY TEST (Continued)
PRINCIPAL INVESTIGATOR(S)
Time Added
Test Material/
Daphnids
Test
Chamber Volume
No. of Daphnids
Per Vessel
Total
Solution Volume
No. of Replicates
Per Treatment
Level
Type
Test
Vessel
Solution
Volume Per
Replicate
Test Vessel
Age of
Daphnid
at Test
Initiation
(Hours)
Water Quality of Dilution Water
Data Transcribed
Notebook
Page No.
Location
Source
Batch No.
pH
Total
Alkalinity
Total Hardness
Conductivity
Comments
NO
Effect Level Through 48 Hours
OBSERVATION KEY
OS - On Surface CO -
OB - On Bottom CLDY -
LETH - Lethargic PRE -
ERR - Erratic Swimming UM -
FC - Flared Carapace PM -
SC - Swimming, Carrying F •
Caught On
Cloudy
Precipitate
Undissolved
Particulate
Film
Material
Matter
SIGNATURE INITIALS
37
-------
FORM
CHRONIC TOXICITY TEST
Test Material
Sponsor
Chronic Toxlclty Test
D.
Treatment Level Principal Investigator! s)
Test
Day
Data
By
Age of D. magna
magna Sourci
s Brood No. Removed From
Photoperlod/Ught Intensity
Number alive/Number offspring
A'
B
C
0
E
F
G
H
I
J
Soln. Renew
Using D11u.
Wtr. Batch
pH
New/Old
Soln.
DO (mg/L)
New/Old
Soln.
Temp CC)
New/Old
Soln.
CO
CO
(continued)
-------
FORM
CHRONIC TOXICITY TEST (Continued)
OJ
\o
TEST
DAY
DATA
BY
TEST MATERIAL
OBSERVATIONS TREATMENT LEVEL
Total Hardness Total Total Alkalinity Total
Test Sample N of mL Tit rant Hardness Test Sample N or mL Tit rant Alkalinity
Day Size TUrant Used (mg/L CaCOi) Day Size Tltrant Used (»g/L CaC03)
X No. Surviving
SUMMARY
T No. Young/Surviving Adult
7 Total Length of Daphnlds at Day 21
DO Range pH Range Hard. Range Alk. Range
New/Old New/Old New/Old New/Old
Solns. Solns Solns. Solns.
Temperature Range
-------
APPENDIX G
QUALITY CONTROL AND QUALITY ASSURANCE
USE OF STANDARDIZED METHODS
Close adherence to standardized procedures for toxicity testing such as those
provided in this protocol is a first and critical step in affecting reproducible
bioassay data quality. Many quality control features have been incorporated
into the protocol; some of these may be transparent to the user. These features
are integrated into each of the specific procedures for preparation of materials,
food and test organisms, and into the conduct of the testing program.
USE OF GOOD LABORATORY PRACTICE STANDARDS
Laboratories performing toxicology testing should have in place Good
Laboratory Practice (GLP) Standards that apply to all phases of the operation,
i.e., organization, personnel, facilities, equipment, reagents and chemicals,
operating procedures, test and control substances, study plan, records, and
reporting. Appropriate Good Laboratory Practice Standards are described in 40
CFR Part 792, Volume 48, Tuesday, November 29, 1983, pp. 53937-53944.
AUDIT GUIDELINES
To assist laboratory managers, quality assurance officers, or researchers
in evaluating their own operations, a set of guidelines in the form of a detailed
questionnaire has been provided (see Appendix H).
REFERENCE TOXICANT
Another key to establishing and maintaining a system to control the bioassay
operation is the routine use of a reference toxicant(s) to determine the current
responsiveness of the daphnid test system. Routine use of carefully prepared
dilutions of reference toxicant stock solution in 48-hour acute tests provides
an excellent mechanism for determining whether test organism response is within
the expected range (historical control chart limits) for that laboratory.
Sodium pentachlorophenate has been shown in a number of EPA-sponsored
studies to provide highly reproducible toxicity to Daphnia magna. Well
characterized solutions of sodium pentachlorophenate will be available on a
continuing basis from the Environmental Monitoring and Support Laboratory
(EMSL), Cincinnati, Ohio. These solutions will be suitable for dilution to
test concentrations and are stable for prolonged periods if maintained in
accordance with instructions provided with the stock solutions. For
40
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information on obtaining reference toxicant solutions, call Cornelius Weber
at (513) 569-7337, EMSL-CIN, or Llewellyn Williams at (702) 798-2138, EMSL-LV.
"STANDARD" TROUT FOOD
Trout food suitable for use with this protocol is stocked by the ERL-
Duluth and is available upon request. A batch is maintained, and portions will
be provided from that source so long as the potency of the nutrients is retained,
and no deleterious changes (e.g., excessive bacterial or fungal contamination)
are noted in the material. The potency and contamination checks will be based
upon periodic analyses of the material and on continued performance checks to
determine that negative and positive (reference-toxicant-tested) controls are
within laboratory historical control limits. In advance of the time that the
replacement of the master stock of trout chow at ERL-Duluth is required,
additional batches will be procured and tested to assure that they conform with
the basic specifications provided by the Fish and Wildlife Service and that
they meet performance specifications then current at the ERL-Duluth. The batch
selected will be blended and will be properly maintained for future availability
and use.
COOPERATIVE ACTIVITIES
Laboratories are urged to cooperate with one another in mutual evaluation
of testing performance through such mechanisms as split-sample exchanges to
compare system performance on identical materials or independent processing of
one another's data sets to assure uniformity in test data interpretation.
Regular programs established in recognition of the potential mutual benefits
are likely to improve the comparability of data among testing laboratories and
to improve the overall quality of the resulting data.
EPA TECHNICAL SUPPORT
Under various EPA programs, occasional test samples (natural, spiked, or
synthetic matrices) become available from the EMSLs for distribution to
laboratories willing to participate in round-robin performance evaluation
studies. Involvement in such a program can be very beneficial to the
laboratories involved; each participant has an opportunity to evaluate the
performance of his laboratory against that of others using the same procedures.
Based on historical precedent, it is expected that the laboratories of the
Office of Research and Development under the aegis of the EPA will continue to
provide technical assistance to those testing and contract laboratories attempt-
ing to improve their bioassay testing performance by using EPA methods. Call
the authors for further information.
41
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APPENDIX H
STANDARD OPERATING PROCEDURE (SOP) FOR
AUDITING DAPHNIA ACUTE EC50 TEST
The following is an audit guideline in the form of a questionnaire
prepared by an expert panel knowledgeable in aquatic toxicity testing and
quality assurance. This questionnaire may be used by an organizational quality
assurance officer to supplement GLP or QA systems audits of a testing operation
or may be used effectively by individual investigators to check critical ele-
ments of their own performance. It is intended to go beyond the scope of
routine GLP audits. Although the SOP is highly specific for the Daphnia Acute
EC5Q Test, many of the questionnaire elements apply as well to Daphnia static
renewal tests used to estimate chronic toxicity.
42
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AUDIT SOP FOR DAPHNIA ACUTE EC50 TEST
Section I. Basic Study Information
When possible, the following information should be obtained in advance of the
audit being scheduled:
A. Auditor Information:
1. Names(s)/Affiliation:
2. Date of Audit: , 19_
B. Testing Lab Information:
1. Laboratory Name:
2. Laboratory Address:
3. Laboratory Phone No.: ( )
4. Laboratory Contact: Dr./Mr./Ms.
5. Principal Investigators:
6. Compound Code or Chemical Tested:
7. Physical Description of Compound or Chemical Tested:
8. Type of Study:
C. Sponsor Information:
1. Sponsor Name:
2. Sponsor Address:
3. Sponsor Contact:
4. Sponsor Phone No.: ( )
43
-------
II. Water
*1. Was the same water used for cultur-
ing as for dilution Yes No
*2. What was the source of dilution
water used?
Reconstituted. If reconstituted,
was the Marking and Dawson (ASTM)
standard used?
Yes No. If No, cite
reference of other standard used
under comments.
Were reagent-grade chemicals
used to prepare the reconstituted
water? Yes No
Was glass-distilled or carbon-
filtered deionized water with a
conductivity less than microohm/cm
used to prepare the reconstituted
dilution water?
Yes No
Well water
Surface water
Dechlorinated tap water
(specify method of dechlorination
under comments).
If dechlorinated tap water was used,
specify the water chemical parameters
tested and concentrations measured.
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments;
Comments:
Parameter
Concentration
Particulate matter
Total organic carbon
Chemical oxygen demand
Un-ionized ammonia
Residual chlorine
*Asterisked questions are essential and must be addressed during the audit.
44
-------
II. Water - Continued
Total organophos-
phorous pesticides
Total organochlorine
pesticides
Polychlorinated
biphenyls (PCBs)
Organic chlorine
Augmented water
Other (identify under comments)
*3. What was the hardness of the
dilution water (ppm)?
<50 50 to 10
_ >100
III. Daphnia Cultun'ng
*1. What was the average age of the
females when their first broods
were released? days
*2. What was the reproductive rate of
the daphnids for the seven-day
period prior to testing?
young/adult/day (after
release of first brood)
*3. What percentage of the culture stock
died during the 48-hour period prior
to testing? %
*4. Were ephippia produced in the culture?
Yes No
*5. What was the initial source of the
Daphnia stock? (identify under
comments)
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
*Asterisked questions are essential and must be addressed during the audit,
45
-------
III. Daphnla Culturing - Continued
*6. How was.species identification
confirmed?
Taxonomic key (cite reference
under comments)
Specialist confirmation
(identify under comments)
Other (specify under comments)
*7. When was the identification of test
species last confirmed?
Date: /__/
*8. What food was used to feed culture
stock?
Algae
Synthetic (specify source and
batch number under comments)
Combination (algae and synthetic
Other (specify under comments)
Identify under comments, where
applicable, the food organism and
source for each food type checked.
IV. Test Setup: General Procedures
*1. Were glass containers used?
Yes No. If No, explain and
identify container construction
material under comments.
2. How was equipment that came into
contact with the test solutions,
test substance or Daphnia cleaned?
Comments:
Comments:
Comments:
Comments:
Comments:
Phosphate- Acetone Acid Distilled Dilution
Equipment Pre-Cleaned Free Detergent Rinse Rinse Water Water
Rinse Rinse
Exposure chambers
Pipettes/glass tubing
General glassware
Food containers
*Asterisked questions are essential and must be addressed during the audit.
46
-------
III. Daphnia Culturing - Continued
Test probes
Other equipment (specify
under comments)
IV. Test Setup: General Procedures - Continued
Question No. 2. - Continued
Comments:
What was the mean of the test
temperatures over the entire
testing period? C
4. Were the following acclimation and
test conditions the same as the
culture conditions?
Comments:
Comments:
Condition
Yes No
Photoperiod
Temperature
Light Intensity
Light Source
(Specify light source under comments.)
If No, identify the difference.
5. What was the test photoperiod?
hr light
hr dark
*6. How was the test chemical added to
the dilution water?
Direct addition
Comments:
Comments:
Stock solution (if stock solution
was used, answer question 7)
*Asterisked questions are essential and must be addressed during the audit,
47
-------
IV. Test Setup: General Procedures - Continued
*7. How was the stock solution prepared? Comments:
Deionized water
Distilled water
Dilution water
Solvent (specify under comments).
If a solvent was used, answer
question 8-10.
8. What was the highest concentration of
solvent used in the test solution?
PP»n
9. Was the concentration of solvent in
the solvent controls equal to the
highest concentration used in the
test? Yes No
10. Did the concentration of solvent
differ among exposure chambers?
Yes No
11. Were no more than two Daphnia added
to each exposure chamber at one time
and placed into each exposure chamber
sequentially? Yes No
12. What was the loading rate used?
mL/Daphnia
13. Were the exposure chambers placed
in the testing area in a random
manner? Yes No
V. Test Setup: Definitive Test
*1. What was the basis for selection of
the test concentrations?
Range finding test
Limit of water solubility
Upper regulatory limit (cite
document under comments)
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
*Asterisked questions are essential and must be addressed during the audit.
48
-------
Test Setup: Definitive Test - Continued
Other (specify under comments)
What test concentrations and controls
were used? (identify under comments)
For each replicate test concentration
were separate stock solutions and
dilutions prepared? Yes No
Were a minimum of 20 Daphnia exposed
to each test concentration?
Yes No
If No, how many were exposed to each
test concentration?
What was the number of replicate ex-
posure chambers for each test
concentration?
Did each replicate contain the same
number of daphnids?
Yes No
Did all Daphnia used in the test
come from the same stock culture?
Yes No
Was the test begun with Daphnia
<24 hours old? Yes No
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
*9. From what brood were the test organisms Comments:
taken? (specify under comments)
*10. Were immobilized Daphnia left in
the exposure chamber for the
duration of the test?
Yes No
Comments:
11. Note which of the following water quality parameters were measured in
the test solution and at what stage in the test.
Parameter
Dissolved Oxygen
Temperature
Test
Beginning
Middle
of Test
Test
End
All Selective
Concen- Concen-
trations trations
*Asterisked questions are essential and must be addressed during the audit.
49
-------
V. Definitive Test - Continued
pH _
Specific conductance
Comments:
*12. Were any of the following observed during the test?
Parameter
Test
Beginning
Middle
of Test
Surface film
Precipitate
Phase separation
Cloudiness
Adsorption to
exposure chamber
walls
Other (specify under
comments)
Comments:
Test
End
All Selective
Concen- Concen-
trations trations
13. What was the lowest dissolved oxygen
level measured, expressed as percent
saturation? % saturation
Comments;
VI. Analytical Measurements
1. Were expected test concentrations
conf i rmed? _ Yes _ No
*2. Were standard chemical analytical
techniques used to confirm concen-
trations? _ Yes _ No (if Yes,
cite reference(s) under comments)
3. Were expected test concentrations con-
firmed by methods other than standard
chemical analyses? _ Yes _ No
(if Yes, identify method under comments)
Comments:
Comments:
Comments:
*Asterisked questions are essential and must be addressed during the audit.
50
-------
VI.
4.
5.
Analytical Measurements - Continued
Identify under comments the limit of
quantitation of the procedure.
When were the concentrations confirmed
and at what test concentrations?
Time
Concentrations
Test beginning
Middle of test
Test end
6. How many replicate chambers per con-
centration were analyzed?
What fractions of the test solution
were analyzed?
Dissolved
Particulate
Total
*8. Did the measured concentration of test
substance among the replicate exposure
chambers in any one test concentration
at any given time vary more than 20
percent? Yes No
9. Specify under comments the highest and
lowest percent change in the measured
test concentration in each exposure
chamber from the beginning to the
end of the test.
10. Specify under comments the highest
and lowest mean percent difference
between expected and measured test
concentrations at time zero.
VII. Data Analysis
1. At what time during the test were
observations of the exposure chambers
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
*Asterisked questions are essential and must be addressed during the audit.
51
-------
VII. Data Analysis - Continued
2.
*3.
4.
7.
8.
9.
made for 'immobilization and abnormal
behavior?
Were greater than 10 percent of either
dilution water or solvent controls
immobilized? _ Yes _ No
What criteria were used to determine
immobilization? (discuss under
comments)
Were Daphnia physically injured during
handling included in the data analysis?
_ Yes _ No
Were partial mortalities on each side
of the EC5Q observed? _ Yes _ No
Comments:
Comments:
Comments:
Comments:
Comments:
Comments:
Were 24- and 48-hour ECsg response Comments:
curves and slopes developed for the
immobilization data? Yes No
Were concentration-response curves
and slopes developed for the immo-
bilization data? Yes No
Was a statistical test of goodness-
of-fit performed on the concentration-
response curves? Yes No
Identify under comments the statistical
method and reference used to calculate
24- and 48-hour ECsg values and their
respective 95 percent confidence limits. ZHZZZZHZH
VIII. Reporting
The final report contains which of the following information:
Comments:
Name of test(s)
Name of sponsor
Testing laboratory
Name of study di rector
Principle investigator
Testing dates
Comments:
*Asterisked questions are essential and must be addressed during the audit,
52
-------
VIII. Reporting - Continued
Detailed test chemical description Comments:
including:
Source
Lot number
Composition (identify and note
concentration of major ingredients
and impurities)
Physical/chemical properties Comments:
Carriers or other additives and their
concentrations
Dilution water source Comments:
Chemical characteristics of dilution
water (e.g., conductivity, hardness, pH,
etc.)
Description of any pretreatment
Detailed information on daphnids used Comments:
as brood stock including:
Scientific name
Method of verification
Culture method
Test chamber description Comments:
Volume of solution to chambers
Description of test start-up (e.g.,
conditioning, test chemical additions)
Test organisms per test chamber
Test organism age
Replicates per treatment
Concentration of test chemical in each Comments:
exposure chamber
Percent or number of organisms
immobilized in each exposure chamber at
each observation period
Concentration-response curves
Goodness-of-fit test results
24- and 48-hour ECso values and 95
percent confidence limits
Method of calculating ECso values
53
-------
VIII. Reporting - Continued
Results of water quality analyses Comments:
including:
Methods ZZZZI
Method validations
Reagent blanks
Spikes
Results of test chemical concentrations Comments:
including:
Methods ^^^
Method validations
Reagent blanks
Spikes
Data records of culture, acclimation, Comments:
and test temperatures.
Data records of culture, acclimation, Comments:
and test lighting.
54
-------
AUDIT SUMMARY
55
-------
CERTIFICATION OF INFORMATION PROVIDED
I have reviewed the completed audit form and have found the information
provided to be true and accurate to the best of my knowledge.
Signature of authorized representative of laboratory being audited Date
Signature of auditor or audit team leader Date
56
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GLOSSARY FOR AUDIT SOP
1. Reconstituted Water - Any water where reagent grade chemicals are added to
distilled or deionized water.
2. Augmented Water - Any water where reagent-grade chemicals are added to
natural waters.
3. Random - Placement of organisms in the exposure chambers and placement of
exposure chambers to avoid systematic bias such as differences in light
intensity, temperature, swimming ability, etc., of Daphnia.
57
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