£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

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                  FORM	

          STATIC ACUTE TOXICITY TEST

                              TEST MATERIAL
Date


Time


Control
Temp


Data By


  REP
CONC
DO
PH
HRD
ALK
DO
HRD
ALK
                                                    (continued)
                       34

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              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

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                                                               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

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                                                        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

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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

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 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

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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

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 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

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 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

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    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

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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

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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

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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

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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

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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

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AUDIT SUMMARY
      55

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                    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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