United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
EPA 712-C-96-132
April 1996
&EPA Ecological Effects Test
OPPTS 850.1800
Subchronic Toxicity Test

Public Draft"

This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP, or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and

OPPTS 850.1800 Tadpole/sediment subchronic toxicity test.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.1995 Tadpole/sediment
subchronic toxicity test.
(b)	Purpose. This guideline may be used to develop data on the
subchronic toxicity of chemical substances and mixtures subject to envi-
ronmental effects testing. This guideline prescribes tests to be used to de-
velop data on the subchronic toxicity of chemicals sorbed to natural sedi-
ments to bullfrog tadpoles. The EPA will use data from these tests in as-
sessing the hazard of a chemical to the environment.
(c)	Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA), and the definitions in 40 CFR part 792—Good Lab-
oratory Practice Standards for physical, chemical, persistence, and ecologi-
cal effects testing apply to this test guideline. The following definitions
also apply:
Acclimation means the physiological compensation by test organisms
to new environmental conditions (e.g., temperature, hardness, pH).
Carrier means a solvent or dispersant used to dissolve a test sub-
Cation exchange capacity (CEC) means the sum total of exchangeable
cations that a sediment can absorb. The CEC is expressed in
milliequivalents of negative charge per 100 g or milliequivalents of nega-
tive charge per gram of sediment (dry weight).
Clay mineral analysis means the estimation or determination of the
kinds of clay-size minerals and the amount present in a sediment.
Conditioning means the exposure of construction materials, test cham-
bers, and testing apparatus to dilution water or to test solutions prior to
the start of a test in order to minimize the sorption of the test substance
onto the test facilities or the leaching of substances from the test facilities
into the dilution water or test solution.
Control means the exposure of test organisms to uncontaminated sedi-
Death means the total lack of movement by a test tadpole.
EC50 means that test substance concentration calculated from experi-
mentally-derived growth or sublethal effects data that has affected 50 per-

cent of a test population during continuous exposure over a specified pe-
riod of time.
Flow-through means a continuous or an intermittent passage of dilu-
tion water through a test chamber, or a holding or acclimation tank with
no recycling.
LC50 means the test substance concentration calculated from experi-
mentally-derived mortality data that is lethal to 50 percent of a test popu-
lation during continuous exposure over a specified period of time.
Loading means the ratio of tadpole biomass (in grams, wet weight)
to the volume (in liters) of test solution in a test chamber or passing
through it in a 24-h period.
Lowest-observed-effect-concentration (LOEC) means the lowest treat-
ment (i.e., test concentration) of a test substance that is statistically dif-
ferent in adverse effect on a specific population of test organisms from
that observed in controls.
No-observed-effect-concentration (NOEC) means the highest treat-
ment (i.e., test concentration) of a test substance that shows no statistical
difference in adverse effect on a specific population of test organisms from
that observed in controls.
Organic matter is the organic fraction of the sediment; it includes
plant and animal residues at various stages of decomposition, cells and
tissues of sediment-based organisms, and substances synthesized by the
microbial community.
Particle size analysis is the determination of various amounts of dif-
ferent particle sizes in a sample (i.e., sand, silt, and clay), usually by sedi-
mentation, sieving, micrometry, or combinations of these methods. The
names and diameter ranges commonly used in the United States are pro-
vided in the following Table 1.:
Table 1.—Particle Size
Diameter range
Very coarse sand 	
2.0 to 1.0 mm
Coarse sand 	
1.0 to 0.5 mm
Medium sand 	
0.5 to 0.25 mm
Fine sand 	
0.25 to 0.125 mm
Very fine sand 	
0.125 to 0.052 mm
0.052 to 0.002 mm
< 0.002 mm
Sediment is the unconsolidated inorganic and organic material that
is suspended in and being transported by surface water, or has settled out
and has deposited into beds.

Static means the test solution is not renewed during the period of
the test.
Subchronic toxicity test means a method used to determine the con-
centration of a substance that produces adverse effects on a specified per-
centage of test organisms in a specified period of time (e.g., 30 days)
which is a significant portion of the organism's life cycle. In this guideline,
survival (i.e., death) and growth is used as the measure of toxicity.
Test slurry means the test substance and the natural sediment on
which the test substance is sorbed. This sediment/test substance slurry is
dosed directly into the tadpole.
(d) Test procedures—(1) Summary of the test, (i) Test chambers
are filled with appropriate volumes of dilution water, or appropriate
amounts of contaminated natural sediments and dilution water. If a flow-
through test is performed, the flow of dilution water through each chamber
is adjusted to the rate desired.
(ii)	This toxicity test may be performed by either of two methods:
(A)	Dosing the tadpole directly with a sediment/test substance slurry
and maintaining tadpoles in test chambers with only clean dilution water.
(B)	Maintaining tadpoles in test chambers containing contaminated
sediments and allowing tadpoles to ingest contaminated sediments ad lib-
(iii)	Tadpoles which have been acclimated in accordance with the test
design are introduced into the test and control chambers by stratified ran-
dom assignment.
(iv)	Tadpoles in the test and control chambers should be observed
daily during the test. Dead tadpoles should be removed at least twice each
day and the findings recorded.
(v)	Live tadpoles in the test and control chambers should be weighed
at least every 3 days.
(vi)	The dissolved oxygen (DO) concentration, pH, temperature, and
the concentration of test substance in contaminated sediments and/or water
should be measured at intervals in selected test chambers.
(vii)	Concentration-response curves, LC50, EC50, LOEC, and NOEC
values for the test substance are developed from the survival and growth
data collected during the test.
(2) Range finding test. If the toxicity of the test substance is not
already known, a range-finding test should be performed to determine the
range of concentrations to be used in the definitive test.

(3) Definitive test, (i) This toxicity test may be conducted by either
of two methods:
(A)	Dosing the tadpole directly with a sediment/test substance slurry
and maintaining tadpoles in test chambers with only clean dilution water.
(B)	Maintaining tadpoles in test chambers containing sediments and
allowing tadpoles to ingest contaminated sediments ad libitum.
(ii)	If this test is to be performed by dosing the tadpoles directly,
the sediment/test chemical slurry should be placed directly into the buccal
cavity of the tadpole with a pipet. The slurry should be shaken or mixed
and 50 |iL of the slurry should be placed directly into the posterior portion
of the buccal cavity. The dosed tadpole should be held out of the water
for about 1 minute after dosing to ensure ingestion and then returned to
the test chamber. The test slurry should be prepared by adding 5 mL of
distilled water to 1 g of dry sediment; the test chemical is added and the
final volume is brought to 10 mL. This test slurry should be mixed on
a mechanical shaker for at least 8 h before dosing.
(iii)	If this test is to be conducted by maintaining tadpoles in test
chambers containing contaminated sediments and allowing tadpoles to in-
gest contaminated sediments ad libitum, appropriate amounts of contami-
nated sediments sufficient to cover the bottom of each test chamber with
about 3 to 5 cm of the contaminated sediment should be prepared. An
appropriate amount of clean dilution water (i.e., about 10 to 20 cm above
the sediment) should be added carefully to each chamber followed by tad-
(iv)	It is recommended that this test be performed three times, each
time with a different natural sediment depending on the organic carbon
content: Low (0.1 to 0.2 percent), medium (0.5 to 1.0 percent), and high
(2.0 to 3.0 percent) organic carbon content (refer to OPPTS guideline
835.1220). However, natural sediments with a medium organic carbon
content should be used if this test is to be done only once. Sediments
selected for testing should be characterized by sampling location, general
clay fraction mineralogy, percent sand, silt, and clay (particle size analy-
sis), percent organic matter, percent organic carbon, pH (1:1 solids:water),
and CEC.
(v)	A minimum of 20 tadpoles should be exposed to each of five
or more test substance concentrations (i.e., treatments) and a control. Test
concentrations should be chosen in a geometric series in which the ratio
is between 1.5 and 2.0 mg/kg (e.g., 2, 4, 8, 16, 32, and 64 mg/kg). All
test concentrations should be based on milligrams of test chemical (100
percent active ingredient (AI)) per kilogram of sediment (dry weight). The
concentration range should be selected to determine the concentration-re-
sponse relationship, EC50 values, LOEC, and NOEC values for survival,
sublethal effects, and growth.

(vi)	An equal number of tadpoles should be placed in two or more
replicates. The distribution of individual tadpoles among the test chambers
should be randomized. Test concentrations in sediment and/or dilution
water should be analyzed for test chemical concentrations prior to the start
of the test and at designated times during the test.
(vii)	Every test should include a control consisting of uncontaminated
sediments, the same dilution water, conditions, procedures, and tadpoles
from the same group used in the test, except that none of the test substance
is added.
(viii)	The test duration is 30 days.
(ix)	It is recommended that this test be performed under flow-through
(x)	The number of dead tadpoles should be recorded daily. In addi-
tion, the number of tadpoles showing sublethal effects and the type of
effect (e.g., any abnormal behavior or appearance) should also be recorded
daily. Each tadpole should be weighted every 3 days. Data on survival,
sublethal effects, and growth which are collected during the test are used
to calculate the LC50 value for survival, the EC50 value for sublethal
effects, the EC50 value for growth, and to determine the LOEC and NOEC
values on days 10, 20, and 30.
(xi)	Tadpoles should be fed a suitable food every day. Food which
sinks to the bottom should be used; food which floats on the water surface
should not be used. In tests in which the tadpoles are dosed with a sedi-
ment/test chemical slurry and held in dilution water without sediments,
any excess food or fecal material should be removed when observed. In
tests in which tadpoles are allowed to feed ad libitum on contaminated
sediments, excess food should not be given.
(4) Test results, (i) Survival and growth should be the primary cri-
teria used in this test guideline to evaluate the toxicity of the test sub-
(ii)	In addition to death, any abnormal behavior such as, but not lim-
ited to, erratic swimming, loss of reflex, increased excitability, lethargy,
or any changes in appearance or physiology, such as discoloration (e.g.,
reddened leg), excessive mucous production, opaque eyes, curved spine,
or hemorrhaging should be recorded.
(iii)	Each test and control chamber should be checked for dead or
effected tadpoles and observations recorded every 24 h after the beginning
of the test or within 1 h of the designated times. Dead tadpoles should
be removed at least twice a day.
(iv)	Live tadpoles in the test and control chambers should be weighted
at least every 3 days and fresh weights recorded.

(v)	The mortality data should be used to calculate LC50 values and
their 95 percent confidence limits, and to plot concentration-response
curves at 10, 20, and 30 days. The statistical methods recommended for
use in calculating LC50 values include probit, logit, moving average angle,
and binomial.
(vi)	The sublethal effects and growth (i.e., fresh weight) data should
be used to plot concentration-response curves, calculate EC50 values, and
determine LOEC and NOEC values. The statistical methods recommended
for use in calculating the EC50 values include probit, logit, moving aver-
age angle, and binomial. Appropriate statistical methods (e.g., analysis of
variance and multiple comparison test) should be used to test for signifi-
cant differences between treatment means and determine LOEC and NOEC
(vii)	A test is unacceptable if:
(A)	More than 20 percent of the control tadpoles die or appear to
be stressed, or are seen to be diseased during the test.
(B)	The tadpoles in the control lose a significant amount of weight
during the test, i.e. 30 percent.
(5) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, total organic carbon (TOC)
or chemical oxygen demand (COD), and particulate matter of the dilution
water should be measured in the control test chambers at the beginning
of each static test and at the beginning and end of each flow-through test.
The month-to-month variation of the above values should be less than 10
percent and the pH should vary less than 0.4 units.
(B)	During static tests, the DO concentration, temperature, and pH
should be measured in each test chamber at the beginning of the test, and
as often as needed thereafter, to document changes from the initial levels.
The dilution water volume should not be reduced by more than 10 percent
as a result of these measurements.
(C)	During flow-through tests, the DO, temperature, and pH measure-
ments should be made in each chamber at the beginning of the test and
every 48 hours thereafter until the end of the test. It is recommended that
this test be done under flow-through conditions.
(ii) Collection of samples for measurement of test substance. Sam-
ples of sediment to be analyzed for the test substance should be taken
with a coring device. Samples of dilution water to be analyzed for
desorbed test substance should be taken midway between the top, bottom,
and sides of the test chamber. These samples should not include any sur-
face scum or material dislodged from the bottom or sides. Samples should
be analyzed immediately or handled and stored in a manner which mini-

mizes loss of test substance through microbial degradation,
photodegradation, chemical reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) The concentration of test
substance in sediment and/or dilution water should be measured at a mini-
mum in each test chamber at the beginning (zero-hour, before tadpoles
are added) and every 10 days thereafter.
(B)	The analytical methods used to measure the amount of test sub-
stance in a sample should be validated before beginning the test. The accu-
racy of a method should be verified by a method such as using known
additions. This involves adding a known amount of the test substance to
three samples of dilution water or sediment taken from a chamber contain-
ing dilution water and the same number of tadpoles as are used in the
test. The nominal concentration of the test substance in those samples
should span the concentration range to be used in the test. Validation of
the analytical method should be performed on at least 2 separate days
prior to starting the test.
(C)	An analytical method is not acceptable if likely degradation prod-
ucts of the test substance give positive or negative interferences, unless
it is shown that such degradation products are not present in the test cham-
bers during the test.
(D)	In addition to analyzing samples of dilution water and sediment,
at least one reagent blank, containing all reagents used, should also be
(E)	Among replicate test chambers, the measured concentrations in
sediment should not vary more than 20 percent. The measured concentra-
tion of the test substance in sediment in any chamber during the test should
not vary more than 30 percent from the measured concentration prior to
initiation of the test.
(F)	The mean measured concentration of test substance in sediment
(dry weight) should be used to plot all concentration-response curves and
to calculate all LC50, EC50, LOEC, and NOEC values.
(e) Test conditions—(1) Test species—(i) Selection. The test species
for this test is the bullfrog tadpole (Rana catesbeiana).
(ii) Age and condition of tadpoles. (A) Tadpoles having the mor-
phological characteristics of premetamorphic stages VI through IX as de-
scribed by Taylor and Kollros (1946) under paragraph (g)(3) of this guide-
line, characterized by the emergence of hind paddles and respiration by
gills, should be used. Tadpoles used in a test should be the same age,
weight (i.e., 2 to 5 g), and be of normal size and appearance for their
age. The longest tadpole should not be more than twice the length of the
shortest tadpole.

(B)	All newly acquired tadpoles should be quarantined and observed
for at least 14 days prior to use in a test.
(C)	Tadpoles should not be used for a test if they appear stressed
or if more than 5 percent die during the 48 h immediately prior to the
(iii) Acclimation of test tadpoles. (A) If the holding water is not
from the same source as the test dilution water, acclimation to the dilution
water should be done gradually over a 48-h period and tadpoles should
be held an additional 14 days in the dilution water prior to testing. Any
changes in water temperature should not exceed about 1 °C per hour or
3 °C per day. Tadpoles should be held for a minimum of 7 days at the
test temperature prior to testing.
(B) During the final 48 h of acclimation, tadpoles should be main-
tained in facilities with background colors and light intensities similar to
those of the testing area.
(2) Facilities—(i) General. Facilities needed to perform this test in-
(A)	Flow-through tanks for holding and acclimating tadpoles.
(B)	A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.
(C)	Apparatus for straining particulate matter, removing gas bubbles,
or insufficiently dissolved oxygen, respectively.
(D)	Apparatus for providing a 16-h light/8-h dark photoperiod with
a 15- to 30-min transition period.
(E)	Chambers for exposing test tadpoles to the test substance.
(F)	A dilution water delivery system for flow-through tests.
(ii) Construction materials. Construction materials and commer-
cially purchased equipment that may contact the stock solution or dilution
water should not contain substances that can be leached or dissolved into
aqueous solutions in quantities that can alter the test results. Materials and
equipment that contact stock or test solutions should be chosen to mini-
mize sorption of test chemicals. Glass, no. 316 stainless steel, and
perfluorocarbon plastic should be used whenever possible. Concrete, fiber-
glass, or plastic (e.g., PVC) may be used for holding tanks, acclimation
tanks, and water supply systems, but they should be thoroughly condi-
tioned before use. If cast iron pipe is used in freshwater supply systems,
colloidal iron may leach into the dilution water and strainers should be
used to remove rust particles. Rubber, copper, brass, galvanized metal,

epoxy glues, and lead should not come in contact with the dilution water
or stock solution.
(iii)	Dilution water delivery system. In flow-through tests, the sys-
tem used should be calibrated before each test. Calibration includes deter-
mining the flow rate of dilution water through each chamber. The general
operation of the dilution water delivery system should be checked twice
daily during a test. The 24-h flow rate through a test chamber should
be a minimum of six tank volumes. During a test, the flow rates should
not vary more than 10 percent from one test chamber to another or from
one time to any other.
(iv)	Test chambers. Test chambers made of stainless steel should
be welded, not soldered. Test chambers made of glass should be fused
or bonded using clear silicone adhesive. As little adhesive as possible
should be left exposed in the interior of the chamber.
(v)	Cleaning of test system. Dilution water delivery systems and test
chambers should be cleaned before each test. They should be washed with
detergent and rinsed in sequence with clean water, pesticide-free acetone,
clean water, and 5 percent nitric acid, followed by two or more changes
of dilution water.
(vi)	Dilution water. (A) Clean surface or ground water, reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
test tadpoles will survive in it for the duration of the holding, acclimating,
and testing periods without showing signs of stress, such as discoloration
(i.e., reddened leg), hemorrhaging, disorientation, or other unusual behav-
ior. The quality of the dilution water should be constant and should meet
the specifications in the following Table 2. when analyzed (at least twice
a year).
Table 2.—Specifications for Dilution Water
Particulate matter 		20.0 mg/L
Total organic carbon (TOC) 		20 mg/L
Chemical oxygen demand (COD) 		5.0 mg/L
Un-ionized ammonia		1.0 ^g/L
Residual chlorine 		1.0 ^g/L
Total organochlorine pesticides		50.0 ng/L
Total organochlorine pesticides.
plus polychlorinated biphenyls (PCBs) 		50.0 ng/L
Organic chlorine 		25.0 ng/L
(B) The concentration of DO in the dilution water should be between
90 and 100 percent saturation, or >5 mg/L at sea level. If necessary, the
dilution water can be aerated before the addition of the test substance.

All reconstituted water should be aerated before use. Hardness should be
<180 mg/L as CaCOs; pH should be 6.5 to 8.5.
(C)	If disease organisms (e.g., pathogenic bacteria) are present in the
dilution water in sufficient numbers to cause infection, they should be
killed or removed by suitable equipment.
(D)	Glass distilled or carbon filtered deionized water with a con-
ductivity less than 1 (iS/cm is acceptable for use in making reconstituted
water. If the reconstituted water is prepared from a ground or surface water
source, conductivity, and TOC or COD should be measured on each batch.
(vii) Carriers. (A) Distilled water should be used in making stock
solutions of the test substance. If a carrier is absolutely necessary to dis-
solve the test substance, the volume used should be minimal. If the test
substance is a mixture, formulation, or commercial product, none of the
ingredients is considered a carrier unless an extra amount is used to pre-
pare the stock solution. Concentrations of stock solution should be based
on 100 percent AI of the test chemical.
(B) Triethylene glycol and dimethyl formamide are the preferred car-
riers, but acetone can also be used.
(3) Test parameters—(i) Loading. The number of tadpoles placed
in a test chamber should not be so great as to affect the results of the
test. The loading should not be so great that the test substance concentra-
tions in treated sediments are decreased by more than 20 percent due to
uptake by the tadpoles. Loading should not exceed one tadpole per liter
of dilution water in the test chamber at any time. Loading rates should
be adjusted to maintain the DO concentration above the recommended lev-
els and the ammonia concentration below 20 (ig/L.
(ii)	Dissolved oxygen concentration. The DO in each test chamber
should be greater than 5.0 mg/L.
(iii)	Temperature. The test temperature should be about 18 °C. The
temperature should be measured at least hourly in one test chamber.
(iv)	Light. A 16-h light/8-h dark photoperiod with a 15- to 30-
minute transition period should be maintained.
(e) Reporting. (1) The final report should include, but not necessarily
be limited to, the following information.
(i)	Name and address of the facility performing the study, and the
dates on which the study was initiated and was completed, terminated,
or discontinued.
(ii)	Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.

(iii)	Statistical methods used for analyzing the data. A description
of the transformations, calculations, or operations performed on the data,
a summary and analysis of the data, and a statement of the conclusions
drawn from the analysis.
(iv)	The test substance identified by name, Chemical Abstracts Serv-
ice (CAS) registry number or code number, source, lot or batch number,
strength, purity, and composition, or other appropriate characteristics.
(v)	Stability of the test and, if used, control substances under the con-
ditions of administration.
(vi)	A description of the methods used, which should include the fol-
(A)	Description of the test chambers, the depth and volume of solu-
tion in the chamber, the specific way the test was begun (e.g., conditioning
and test substance additions), and for flow-through tests, a description of
the dilution water delivery system including a diagram if the design is
(B)	The source of the dilution water, a description of any
pretreatment, and the measured hardness, acidity, alkalinity, pH, con-
ductivity, TOC or COD, and particulate matter.
(C)	The source of the natural sediment (i.e., sampling location), sedi-
ment physical-chemical properties, percent sand, silt, and clay (particle
size analysis), percent organic matter, percent organic carbon, pH (1:1 sol-
ids: water), CEC, general clay fraction mineralogy, and procedures used
to determine the above properties.
(D)	Methods used to determine the placement of test chambers and
the assignment of treatment concentrations to particular test chambers to
ensure randomization of exposure.
(E)	Frequency, duration, and methods of observations.
(F)	Detailed information about the test tadpoles, including the sci-
entific name and method of verification, source of test species, histories
of the species, average fresh weight (grams), average size, age, observed
diseases, treatments and mortalities, acclimation procedures, and food
(G)	The number of treatments and replicates used, the number of or-
ganisms per replicate, the loading rate, and the flow rate of dilution water
for flow-through tests.
(H)	A description of the preparation of the sediment/test substance
slurry or the treated sediments. A description of the dosing procedures
if tadpoles were dosed directly.

(1)	The concentration of the test substance in the test slurry or in
sediments and/or dilution water in each test chamber just before the start
of the test and at all subsequent sampling periods. The concentration of
the test substance in the stock solution, if used, and the type and concentra-
tion of carrier solvent, if used.
(vii)	The measured DO, pH, and temperature and the lighting regime.
(viii)	The reported results should include:
(A)	The results of the preliminary test and measurements. The number
of tadpoles and concentrations of test substance used and observed effects
on tadpoles should be stated.
(B)	For the definitive test, in each untreated control and for each treat-
ment concentration used:
(7) The number of dead and live tadpoles.
(2)	The percentages of tadpoles that died or showed adverse sublethal
(3)	The number that showed any abnormal effects.
(4)	The fresh weights of live tadpoles.
(5)	The LC50, EC50, LOEC, and NOEC values at days 10, 20, and
Results of the data analysis should include the concentration-response
curves with 95 percent confidence limits and the results of a goodness-
of-fit (e.g., X2-square test).
(ix)	A description of all circumstances that may have affected the
quality or integrity of the data.
(x)	Methods and data records of all chemical analyses of water quality
parameters and test substance concentrations, including method validation
and reagent blanks.
(xi)	The name of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the names of all supervisory
personnel involved in the study.
(xii)	The signed and dated reports of each of the individual scientists
or other professionals involved in the study including each person who,
at the request or direction of the testing facility or sponsor, conducted
an analysis or evaluation of data or specimens from the study after data
generation was completed.
(xiii)	The locations where all specimens, raw data, and the final report
are stored.

(xiv) The quality control statement prepared and signed by the quality
assurance unit.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1)	National Research Council. Amphibians: Guidelines for the
Breeding, Care, and Management of Laboratory Animals. National Acad-
emy of Sciences, Washington, DC (1974).
(2)	Perkins, K. W. et al. Reptiles and Amphibians: Care and Culture.
Carolina Biological Supply Co., Burlington, NC (1981).
(3)	Taylor, A. C. and Kollros, J. J. Stages in the Normal Development
of Ranapipiens. Anatomy Records 94:2 (1946).