SEPA
United States
Environmental Protection
Agency
Office of Pesticides Programs
Washington, DC 20460
EPA 540/9-86-141
July 1966
Hazard Evaluation
Standard Evaluation
Daphnia Magna Life-Cycle (21-Day
Renewal) Chronic Toxicity Test
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is one of a set of
guidance documents which explain the procedures used to evaluate
environmental and human health effects data submitted to the
Office of Pesticide Programs. The SEPs are designed to ensure
comprehensive and consistent treatment of major scientific topics
in these reviews and to provide interpretive policy guidance
where appropriate. The Standard Evaluation Procedures will be
used in conjunction with the appropriate Pesticide Assessment
Guidelines and other Agency Guidelines. While the documents were
developed to explain specifically the principles of scientific
evaluation within the Office of Pesticide Programs, they may also
be used by other offices in the Agency in the evaluation of
studies and scientific data. The Standard Evaluation Procedures
will also serve as valuable internal reference documents and will
inform the public and regulated community of important consider-
ations in the evaluation of test data for determining chemical
hazards. I believe the SEPs will improve both the quality of
science within EPA and, in conjunction with the Pesticide Assess-?
ment Guidelines, will lead to more effective use of both public
and private resources.
in W. Melone, Director
Hazard Evaluation Division
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TABLE OF CONTENTS
I. INTRODUCTION
A. When Required 1
B. Purpose 1
C. Test Material .-....' 2
D. Acceptable Protocols ' 2
II. MATERIALS, METHODS AND REPORTING REQUIREMENTS
A. Biological System 3
1. Test Organisms . .... 3
2. Source 3
3 . Food 3
4. Beginning the Test ,.. 3
5. Renewal 3
6. Durat ion 4
7. Test Rejection 4
8. Data Endpoints •••• 4
B. Physical System 4
1. Test Water 4
2. Temperature 5
3. Photoper iod 5
4. Test Vessels 5
5. Aeration 5
C. Chemical System 5
1. Concentrations 5
2. Measurement of Other Variables 5
3 . Solvents 5
D. Calculations 6
III. REVIEWER'S EVALUATION
A. Verification of Statistical Analysis 6
B. Conclusions . . 6
1. Rationale ... 7
2. Reparability 7
3. Descriptive Conclusions 7
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DAPHNIA MAGNA LIFE-CYCLE (21-DAY RENEWAL)
CHRONIC TOXICITY TEST
I. INTRODUCTION
A. When Required
The Daphnia magna life-cycle studyV is required to support
registration of an end-use pesticide product that is applied
directly to water or expected to be transported to water from
the intended use site, and when any of the following conditions
apply:
0 If the pesticide is intended for use such that its
presence in water is likely to be continuous or
recurrent regardless of toxicity, as revealed by studies
required by 40 CFR § 158.130;
0 If any LC50 or EC50 value determined in testing required
by 40 CFR §158.145 [§§72-1, -2, or -3] is less than 1
mg/1;
0 If the estimated environmental concentration in water
is equal to or greater than 0.01 of any EC50 or LC50
determined iri acute testing for aquatic organisms
required by 40 CFR § 158.145; or
0 If the actual or estimated environmental concentration
in water resulting from use is less than 0.01 of any
EC50 or LC50 determined in acute testing for aquatic
organisms required by 40 CFR 158.145 and any of the
following conditions exist:
Studies of other organisms indicate the
reproductive physiology of invertebrates may be
affected;
The pesticide is persistent in water (e.g., half-
life in water greater than 4 days); or
Physicochemical properties indicate cumulative
effects.
In cases where risk criteria are exceeded for both fish
and aquatic invertebrates, the more sensitive organism must
be tested in a fish early life-stage or invertebrate life
cycle study. Both studies may, however, be required to
complete a risk assessment.
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B. Purpose
0 The Daphnia magna life-cycle study is intended to measure
pesticidal effects on daphnid reproduction, survival
and growth. Survival, adult length, and the average
number of offspring per adult per reproduction day are
measured in this study;
0 Establish chronic toxicity levels of the active
ingredient to daphnids; < .
0 Compare toxicity information with measured or estimated
pesticide residues in an aquatic environment in order
to assess potential impact to aquatic invertebrates;
0 Provide support for precautionary label statements;
0 Indicate the need for further laboratory testing or
field testing; and
° Renewal tests, may not t>e. applicable to chemicals which
have high oxygen demand, are highly volatile, are
transformed, or sorbed to glass. In these cases,
flow-through tests may be more appropriate. .
C. Test Material
Testing must be conducted with the technical grade of the
active ingredient (a.i.). If more than one. active ingredient
constitutes a technical product, the technical grade of each
active ingredient must be tested separately.
D. Acceptable Protocols > :
EEB does not endorse any one protocol. It is sometimes
necessary and desirable to alter the procedures presented in
published protocols to meet the needs of the chemical or test
organisms used. However, EEB does recommend some protocols as
guidance for developing a daphnid life-cycle test. These
protocols include:
Biesinger, K.E, 1974 (a) . Procedure for Daphnia magna
chronic test in standing system. U.S. EPA, Eviron. Res.
Lab., Duluth, MN. Federal Register 40 (123) : 26902-26903
pp. (June 25, 1975}.
American Public Health Association, American Water Works
Association and Water Pollution Control Federation. 1985.
Standard Methods for the Examination of Water and Wastewater.
Sixteenth Edition. Publication Office: American Public
Health Association, 1015 18th Street, N.W., Washington,
D.C. 20036. 765 pp.
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ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and
Amphibians. American Society for Testing and Materials,
1916 Race street, Philadelphia, PA 19103.
II. MATERIALS, METHODS AND REPORTING REQUIREMENTS ,
A. Biological System
1. Test Organisms
Daphnial/ are used in this test because they represent an
important aquatic phyla, they are members of a trophic level of
primary consumers, and they are a sensitive test organism for
pesticide evaluation. The use of these organisms is also
advantageous because of their short life-cycle, ease of culture,
and requirements for limited space and water volume.
2. Source
Daphnia can be obtained from laboratory, commercial, or
wild stocks. All test organisms must be produced from a
laboratory reared culture that has been maintained for at least
21 days at test conditions in dilution water with renewal of
the culture medium at least three times a week. The identity
of organisms must ;be verified regardless of any information
that comes with the organisms.
3. Food
A variety of foods appear to be adequate for culturing
daphnids. These include: 1) synthetic foods (trout chow);
2) synthetic foods in combination with alfalfa yeast and algae;
and 3) algae including Ankistrodesmus falcatus, Chlamydamonas
reinhardtii, and Selenastrum capricornutum. Other food mixtures
may also be acceptable. .
4. Beginning the Test
Prior to starting a test, daphnids which are at least
10-12 days old (those that have had at least one brood) should
be separated from the culture, put in a separate culture
container and maintained for at least 2.1 days to insure that
good health and conditions are present.
V Registrants wishing to use other test species for this study
may do so, provided those species are deemed acceptable by
the Agency.
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Young daphnids _< 24 hours old are obtained from this
subculture and are used to start the test. Ten 250 ml beakers
(200 mis of test solutidn) are used for each toxicant concen-
tration: a) seven beakers at each concentration will contain
one daphnid each for collection of data on survival, growth,
and reproduction; b) three beakers at each concentration will
contain five daphnids each for collectionof data on survival
only (not reproduction or growth). Assignment of daphnids
should be randomized. A test begins when test^organisms are
first placed in the test solution.
5. Renewal
A renewal schedule (i.e!., Monday, Wednesday, Friday) must
be set-up for counting live and dead daphnids. Parent daphnids
in all beakers are counted and transferred to beakers containing
the same toxicant concentration as that from which they were
removed. In the seven beakers containing one parent daphnid
each, the offspring, both live and dead, are counted and
discarded. '
6. Duration
Testing is concluded on the 21st day. On this final day,
the first generation (parent) daphnids are counted and indivi-
dually measured to the nearest 0.01 mm frpm the apex of the
helmet to the base of the spine. The number of young, both
alive and dead, in each beaker are counted.
7. Test Rejection
A test is rejected if the following occurs:
0 30 percent of.specimens in the controls (including
solvent control) die;
0 Daphnids in either control do not produce at least 40
young after 21 days;
0 Production of ephlppia by any of the controls. These
"resting eggs" are capable of withstanding adverse
environmental conditions;
0 Temperature deviation from 20°C exceeds 5°C for more
than 48 hours;
0 Dissolved oxygen drops below 50 percent of saturation
for more than 48 hours; or
0 pH deviates by more than one unit for more than 48 hours,
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8. Data Endpoints
A report of the results of a test must include data on
the survival of first generation daphnids, production of young
by first generation daphnids at various times for each
treatment, and the length of first generation daphnids at the
end of test. :
B. Physical System
1. Test Water
Test water can be supplied from a well or spring provided
that the source is not polluted. Water must be tested for
pesticides, heavy metals, and other possible contaminants.
Hardness of 160 to 180 mg/L as CaC03 and pH of 7.6 to 8.0 is
recommended. If reconstituted water is used, detailed
descriptions of acceptable procedures for preparing dilutent
are found in the protocols by the American Society of Testing
Materials (1980).
2. Temperature
Life-cycle tests with daphnids should be conducted at
20 + 2°C.
3. Photoperiod •
A 16-hour light and 8-hour dark photoperiod should be
provided. Light intensity should be 400 to 800 Lux (37 to 74
footcandles at the surface of the test solutions) and be
provided by wide-spectrum fluorescent lamps.
4. Test Vessels
Any container made of glass, No. 316 stainless steel, or
perfluorocarbon plastics which hold 200 mis of test solution
can be used. The 250 ml borosilicate glass beakers have been
found convenient to use. Test vessels should be covered with
glass plates to prevent evaporation of test solutions.
5. Aeration
Dilution water should be aerated vigorously insuring that
dissolved oxygen concentration will be at or near 90 to 100
percent saturation. Tests tanks chambers should not be
aerated.
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C. Chemical System
1. Concentrations
A minimum of five concentrations of toxicant and a control
(all duplicated) are used in this chronic test. A solvent
control is added if a solvent is utilized. The recommended
concentration of food to be used is 5 mg/L (dry weight) if
trout food or synthetic diet is used, and 108 algal cells per
liter if an algae diet is Used. For each concentration, ah
aliquote of toxicant is adde<^ to the dilutions water/food
mixture and the solution is well mixed. Test solutions should
be made up less than four hours before the test begins.
2. Measurement of Other Variables
Dissolved oxygen must be measured at each concentration at
least once a week. -Freshwater parameters in a control and one
concentration must be analyzed once a week. These parameters
should include pfl, alkalinity, hardness, and conductance.
3. Solvents :
If solvents other than water are necessary, they should be
used sparingly and not to exceed 0.5 mL/L in a static system.
The following solvents are acceptable:
dimethylformamide
triethylene glycol
methanol .
acetone
ethanol
D. Calculations
Data from these toxlcity studies are of two types,
continuous (e.g., length) of dichotomous (e.g. , number hatching
or surviving). In general, Continuous data will be analyzed
using an appropriate analysis of variance (ANOVA) technique
followed by an appropriate multiple comparison test. Dichotomous
data will be analyzed using a 2 x 2 contingency table. All
test results must be accompanied by copies of the original
(raw) data for the reviewer's evaluation. Transcripts of
original raw data are acceptable if they provide all of the
information in the original data set, including comments or
notes provided by the investigator. '
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III. REVIEWER'S EVALUATION
The reviewer should identify each aspect of the reported
procedures and determine if there is any inconsistency with
recommended methodologies. The number,of deviations and their
severity will determine the validity of the study and the
interpretation of the results. : •
A. verification of Statistical Analysis
The reviewer should ensure that an MATC has been properly
derived by recalculating the reported results. If the recalculated
results differ substantially from the submitted results, the
reviewer should note this and attempt to explain the differences.
B. Conclusions : ,
The significance of inconsistencies in the test procedures
must be determined by the reviewer so that the results of the
test can be categorized as to whether they fulfill Part 158
regulations and are useful in performing .a risk assessment.
Categories are described as:
0 Core; All essential information was reported and the
study was performed according to recommended protocols.
Minor inconsistencies with standard methodologies may be
apparent; however, the deviations do not detract from the
study's soundness or intent. Studies within this category
fulfill the basic requirements of current guidelines and
are acceptable for use in a risk assessment.
0 Supplemental: Studies in this category are scienti-
fically sound; however, they were performed under
conditions that deviated substantially from recommended
protocols. Results do not meet guideline requirements;
however, the information may be useful in a risk
assessment.
Some of the conditions that may place a study in a s
upplemental category include:
Unacceptable test species;
Inappropriate test material; or
Deviations from recommended test solution charac-
teristics (variations in DO, temperature, hardness,
and pH can affect toxicological response).
0 Invalid; These studies provide no useful information.
They may be scientifically unsound, or they were
performed under conditions that deviated so significantly
from recommended protocols that the results will not be
useful in a risk assessment.
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Examples of studies placed in this category commonly
include those where the test system was aerated, test
vessels were constructed from materials other than
glass, or there were problems of solubility of volatility
of the test material. Unless acceptable chemical
analyses of actual toxicant concentrations were performed
in studies such as these, the reviewer cannot be sure
that test organisms were actually exposed to nominally
designated concentrations. ;A study.where the test material
was not properly identified can also be invalidated.
1. Rationale
Identify what makes the study supplemental or invalid.
While all deviations from recommended protocol should be noted,
the reviewer is expected to exercise judgment in the area of
study categorization. '
2. Reparability
Indicate whether, the study Jtiay be upgraded or given a
higher validation category if certain conditions are met.
Usually this would involve the registrant submitting more data
about the study. ;
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3. Descriptive Conclusions
• '
The reviewer should indicate what the results were and how
much information can be drawn from them. These results are
useful in a risk assessment. '
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