[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR797.1600]
[Page 121-132]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 797_ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents
Subpart B_Aquatic Guidelines
Sec. 797.1600 Fish early life stage toxicity test.
(a) Purpose. This guideline is intended to be used for assessing the
propensity of chemical substances to produce adverse effects to fish
during the early stages of their growth and development. This guideline
describes the conditions and procedures for the continuous exposure of
several representative species to a chemical substance during egg, fry
and early juvenile life stages. The Environmental Protection Agency
(EPA) will use data from this test in assessing the potential hazard of
the test substance to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in part 792--Good
Laboratory Practice Standards, apply to this section. In addition, the
following definitions are applicable to this specific test guideline:
(1) ``Acclimation'' physiological or behavioral adaptation of
organisms to one or more environmental conditions associated with the
test method (e.g., temperature, hardness, pH).
(2) ``Carrier'' solvent or other agent used to dissolve or improve
the solubility of the test substance in dilution water.
(3) ``Conditioning'' exposure of construction materials, test
chambers, and testing apparatus to dilution water or to the test
solution prior to the start of the test in order to minimize the
sorption of test substance onto the test facilities or the leachig of
substances from test facilities into the dilution water or the test
solution.
(4) ``Control'' an exposure of test organisms to dilution water only
or dilution water containing the test solvent or carrier (no toxic agent
is intentionally or inadvertently added).
(5) ``Dilution water'' the water used to produce the flow-through
conditions of the test to which the test substance is added and to which
the test species is exposed.
(6) ``Early life stage toxicity test'' a test to determine the
minimum concentration of a substance which produces a statistically
significant observable effect on hatching, survival, development and/or
growth of a fish species continuously exposed during the period of their
early development.
(7) ``Embryo cup'' a small glass jar or similar container with a
screened bottom in which the embryos of some species (i.e., minnow) are
placed during the incubation period and which is normally oscillated to
ensure a flow of water through the cup.
(8) ``Flow through'' refers to the continuous or very frequent
passage of fresh test solution through a test chamber with no recycling.
(9) ``Hardness'' the total concentration of the calcium and
magnesium ions in water expressed as calcium carbonate (mg
CaCO3/liter).
(10) ``Loading'' the ratio of biomass (grams of fish, wet weight) to
the volume (liters) of test solution passing through the test chamber
during a specific interval (normally a 24-hr. period).
(11) ``No observed effect concentration (NOEC)'' the highest tested
concentration in an acceptable early life stage test: (i) which did not
cause the occurrence of any specified adverse effect (statistically
different from the control at the 95 percent level); and (ii) below
which no tested concentration caused such an occurrence.
(12) ``Observed effect concentration (OEC)'' the lowest tested
concentration in an acceptable early life stage test: (i) Which caused
the occurrence of any specified adverse effect (statistically different
from the control at the 95 percent level); and (ii) above which all
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tested concentrations caused such an occurrence.
(13) ``Replicate'' two or more duplicate tests, samples, organisms,
concentrations, or exposure chambers.
(14) ``Stock solution'' the source of the test solution prepared by
dissolving the test substance in dilution water or a carrier which is
then added to dilution water at a specified, selected concentration by
means of the test substance delivery system.
(15) ``Test chamber'' the individual containers in which test
organisms are maintained during exposure to test solution.
(16) ``Test solution'' dilution water with a test substance
dissolved or suspended in it.
(17) ``Test substance'' the specific form of a chemical substance or
mixture that is used to develop data.
(c) Test Procedures--(1) Summary of test. (i) The early life stage
toxicity test with fish involves exposure of newly fertilized embryos to
various concentrations of a test substance. Exposure continues for 28
days post hatch for the minnows and 60 days post hatch for the trout
species. During this time various observations and measurements are made
in a specific manner and schedule in order to determine the lowest
effect and highest no-effect concentrations of the test substance.
(ii) A minimum of five exposure (treatment) concentrations of a test
substance and one control are required to conduct an early life stage
toxicity test. The concentration of the test substance in each treatment
is usually 50 percent of that in the next higher treatment level.
(iii) For each exposure concentration of the test substance and for
each control (i.e., regular control and carrier control is required)
there shall be:
(A) At least two replicate test chambers, each containing one or
more embryo incubation trays or cups; and there shall be no water
connections between the replicate test chambers;
(B) At least 60 embryos divided equally in such a manner that test
results show no significant bias from the distributions, between the
embryo incubation trays or cups for each test concentration and control
(i.e., 30 per embryo cup with 2 replicates);
(C) All surviving larvae divided equally between the test chambers
for each test concentration and control (e.g., 30 larvae per test
chamber with 2 replicates).
(iv) Duration. (A) For fathead minnow and sheepshead minnow a test
begins when the newly fertilized minnow embryos (less than 48-hours old)
are placed in the embryo cups and are exposed to the test solution
concentrations. The test terminates following 28 days of post-hatch
exposure, i.e., 28 days after the newly hatched fry are transferred from
the embryo cups into the test chambers.
(B) For brook trout and rainbow trout a test begins when newly
fertilized trout embryos (less than 96-hours old) are placed in the
embryo trays or cups and are exposed to the test solution
concentrations. The test terminates following 60 days of post-hatch
exposure (for an approximate total exposure period of 90 days).
(C) For silverside a test begins with newly fertilized embryos (less
than or equal to 48 hours old) and is terminated 28 days after hatching.
The chorionic fibrils should be cut before randomly placing the embryos
in the egg incubation cups.
(2) [Reserved]
(3) Range-finding test. (i) A range finding test is normally
performed with the test substance to determine the test concentrations
to be used in the early life stage toxicity test, especially when the
toxicity is unknown. It is recommended that the test substance
concentrations be selected based on information gained from a 4- to 10-
day flow-through toxicity test with juveniles of the selected test
species.
(ii) The highest concentration selected for the early life stage
toxicity test should approximate the lowest concentration indicated in
any previous testing to cause a significant reduction in survival. The
range of concentrations selected is expected to include both observed
effect and no-observed effect levels. The dilution factor between
concentrations is normally 0.50, however, other dilution factors may be
used as necessary.
(4) Definitive test--(i) General. (A) A test shall not be initiated
until after the test conditions have been met and
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the test substance delivery system has been observed functioning
properly for 48-hours. This includes temperature stability, flow
requirements of dilution water, lighting requirements, and the function
of strainers and air traps included in the water-supply system, and
other conditions as specified previously.
(B) New holding and test facilities should be tested with sensitive
organisms (i.e., juvenile test species or daphnids) before use to assure
that the facilities or substances possibly leaching from the equipment
will not adversely affect the test organisms during an actual test.
(C) Embryos should be acclimated for as long as practical to the
test temperature and dilution water prior to the initiation of the test.
(D) When embryos are received from an outside culture source (i.e.,
rainbow and brook trout) at a temperature at variance with the
recommended test temperature they shall be acclimated to the test
temperature. When eggs are received, they should be immediately unpacked
and the temperature of the surrounding water determined. Sudden
temperature changes should be avoided. Acclimation to the appropriate
test temperature should be accomplished within a period of 6 hours, and
should incorporate the use of dilution water.
(E) Embryos should be visually inspected prior to placement in the
embryo cups or screen trays. All dead embryos shall be discarded. Dead
embryos can be discerned by a change in coloration from that of living
embryos (e.g., trout embryos turn white when dead). During visual
inspection, empty shells, opaque embryos, and embryos with fungus or
partial shells attached shall be removed and discarded. If less than 50
percent of the eggs to be used appear to be healthy, all embryos in such
a lot shall be discarded.
(ii) Embryo incubation procedures. (A) Embryos can be distributed to
the embryo cups or screen trays using a pipette with a large bore or a
similar apparatus. Newly-hatched silverside fry are very sensitive to
handling; the egg incubation cups should not be handled at all the first
5 days after hatching begins. Just before hatching is expected to begin,
the embryos should be transferred to clean incubation cups. Trout
embryos can be distributed by using a small container which has been
precalibrated to determine the approximate number of embryos it can
hold; embryos are measured volumetrically in this manner, and are then
poured onto the screen tray (or embryo cup). Trout embryos should be
separated on the screen tray so that they are not in contact with each
other. A final count will ensure the actual number on the screen tray.
After random assignment, the screen trays or embryo cups are placed in
the test chambers.
(B) Each day until hatch the embryos are visually examined. Minnow
embryos may be examined with the aid of a magnifying viewer. Trout
embryos should not be touched. Trout embryos should be maintained in low
intensity light or in darkness until 1-week post hatch, and are usually
examined with the aid of a flashlight or under low intensity light. Dead
embryos should be removed and discarded. Any embryos which are heavily
infected with fungus shall be discarded and shall be subtracted from the
initial number of embryos used as a basis for the calculations of
percentage hatch.
(C) When embryos begin to hatch they should not be handled.
(iii) Initiation of fry exposure. (A) Forty-eight hours after the
first hatch in each treatment level, or when hatching is completed, the
live young fish shall be counted and transferred from each embryo cup
into the appropriate test chamber. For silverside, all surviving fry are
not counted until six days after hatching and are not transferred to
embryo cups. All of the normal and abnormal fry shall be gently released
into the test chamber by allowing the fry to swim out of each embryo
cup; nets shall not be used. The trout embryos incubated on screen trays
will hatch out in the test chambers, therefore handling of fish is not
necessary.
(B) If necessary, fry can be transferred from one replicate embryo
cup to the other replicate within a test concentration to achieve equal
numbers in each replicate chamber.
(C) The number of live fry, live normal fry, live embryos, dead
embryos and unaccounted for embryos for each
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cup shall be recorded when hatching is deemed complete. Those fry which
are visibly (without the use of a dissecting scope or magnifying viewer)
lethargic or grossly abnormal (either in swimming behavior or physical
appearance) shall be counted. Late hatching embryos shall be left in the
embryo cups to determine if they will eventually hatch or not. The range
of time-to-hatch (to the nearest day) for each cup shall be recorded.
(iv) Time to first feeding. (A) The first feeding for the fathead
and sheepshead minnow fry shall begin shortly after transfer of the fry
from the embryo cups to the test chambers. Silversides are fed the first
day after hatch. Trout species initiate feeding at swim-up. The trout
fry shall be fed trout starter mash three times a day ad libitum, with
excess food siphoned off daily. The minnow fry shall be fed live newly-
hatched brine shrimp nauplii (Artemia salina) at least three times a
day.
(B) For the first seven days, feeding shall be done at minimum
intervals of four hours (i.e., 8 am, 12 noon, and 4 pm); thereafter the
fry shall be fed as indicated below.
(v) Feeding. (A) The fathead and sheepshead minnow fry shall be fed
newly hatched brine shrimp nauplii for the duration of the test at
approximately 4-hour intervals three times a day during the week and
twice on the weekend after the first week. Trout fry shall be fed at
similar intervals and may receive live brine shrimp nauplii in addition
to the trout starter food after the first week. Between days 1 and 8
after first hatching, silverside fry are fed the rotifer, Brachionus
plicatilis, three times daily at a concentration of 5,000 to 10,000
organisms per egg cup (based on 15 fish/cup). From days 9 to 11, the fry
shall be fed approximately 2,500 newly hatched brine shrimp (Artemia)
nauplii and 5,000 to 10,000 rotifers twice daily. For the remainder of
the test, the fish will be fed brine shrimp exclusively. The number of
organisms used should be gradually increased to approximately 5,000
nauplii by test day 28.
(B) An identical amount of food should be provided to each chamber.
Fish should be fed ad libitum for 30 minutes with excess food siphoned
off the bottom once daily if necessary.
(C) Fish should not be fed for the last 24 hours prior to
termination of the test.
(vi) Carriers. Water should be used in making up the test stock
solutions. If carriers other than water are absolutely necessary, the
amount used should be the minimum necessary to achieve solution of the
test substance. Triethylene glycol and dimethyl formamide are preferred,
but ethanol and acetone can be used if necessary. Carrier concentrations
selected should be kept constant at all treatment levels.
(vii) Controls. Every test requires a control that consists of the
same dilution water, conditions, procedures, and test organisms from the
same group used in the other test chambers, except that none of the test
substance is added. If a carrier (solvent) is used, a separate carrier
control is required in addition to the regular control. The carrier
control shall be identical to the regular control except that the
highest amount of carrier present in any treatment is added to this
control. 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 prepare the stock solution.
(viii) Randomization. The location of all test chambers within the
test system shall be randomized. A representative sample of the test
embryos should be impartially distributed by adding to each cup or
screen tray no more than 20 percent of the number of embryos to be
placed in each cup or screen tray and repeating the process until each
cup or screen tray contains the specified number of embryos.
Alternatively, the embryos can be assigned by random assignment of a
small group (e.g., 1 to 5) of embryos to each embryo cup or screen tray,
followed by random assignment of a second group of equal number to each
cup or tray, which is continued until the appropriate number of embryos
are contained in each embryo cup or screen tray. The method of
randomization used shall be reported.
(ix) Observations. During the embryo exposure period observations
shall be made to check for mortality. During
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the exposure period of the fry, observations shall be made to check for
mortality and to note the physical appearance and behavior of the young
fish. The biological responses are used in combination with physical and
chemical data in evaluating the overall lethal and sublethal effects of
the test substance. Additional information on the specific methodology
for the data obtained during the test procedure are discussed in the
following sections.
(x) Biological data. (A) Death of embryos shall be recorded daily.
(B) When hatching commences, daily records of the number of embryos
remaining in each embryo cup are required. This information is necessary
to quantify the hatching success. A record of all deformed larvae shall
be kept throughout the entire post-hatch exposure. Time to swim-up shall
be recorded for the trout. Upon transfer of fry from the embryo cups to
the test chambers, daily counts of the number of live fish should be
made. At a minimum, live fish shall be counted on days 4, 11, 18, 25 and
(weekly thereafter for the trout species) finally on termination of the
test.
(C) The criteria for death of young fish is usually immobility,
especially absence of respiratory movement, and lack of reaction to
gentle prodding. Deaths should be recorded daily and dead fish removed
when discovered.
(D) Daily and at termination of the test, the number of fish that
appear (without the use of a magnifying viewer) to be abnormal in
behavior (e.g., swimming erratic or uncoordinated, obviously lethargic,
hyperventilating, or over excited, etc.) or in physical appearance
(e.g., hemorrhaging, producing excessive mucous, or are discolored,
deformed, etc.) shall be recorded and reported in detail.
(E) All physical abnormalities (e.g., stunted bodies, scoliosis,
etc.) shall be photographed and the deformed fish which die, or are
sacrificed at the termination of the test, shall be preserved for
possible future pathological examination.
(F) At termination, all surviving fish shall be measured for growth.
Standard length measurements should be made directly with a caliper, but
may be measured photographically. Measurements shall be made to the
nearest millimeter (0.1 mm is desirable). Weight measurements shall also
be made for each fish alive at termination (wet, blotted dry, and to the
nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish
exposed to the toxicant appear to be edematous compared to control fish,
determination of dry, rather than wet, weight is recommended.
(G) Special physiological, biochemical and histological
investigations on embryos, fry, and juveniles may be deemed appropriate
and shall be performed on a case by case basis.
(5) Test results. (i) Data from toxicity tests are usually either
continuous (e.g. length or weight measurements) or dichotomous (e.g.
number hatching or surviving) in nature. Several methods are available
and acceptable for statistical analysis of data derived from early life
stage toxicity tests; however, the actual statistical methodology to
analyze and interpret the test results shall be reported in detail.
(ii) The significance level for all statistical testing shall be a
minimum of P=0.05 (95 percent confidence level).
(A) Example of statistical analysis. (1) Mortality data for the
embryonic stage, fry stage and for both stages in replicate exposure
chambers should first be analyzed using a two-way analysis of variance
(ANOVA) with interaction model. This analysis will determine if
replicates are significantly different from each other. If a significant
difference between replicates or a significant interaction exists, cause
for the difference should be determined. Modification should then be
made in the test apparatus or in handling procedures for future toxicity
tests. Further calculations should incorporate the separation of
replicates. If no significant difference is observed, replicates may be
pooled in further analyses.
(2) After consideration of replicate responses, mortality data
should then be subjected to one-way ANOVA. The purpose of this analysis
is to determine if a significant difference exists in the percentage
mortality between control fish and those exposed to the test material.
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(3) If the one-way ANOVA results in a F ratio that is significant,
it would be acceptable to perform t-tests on the control versus each
concentration. A second technique is to identify treatment means that
are significantly different; this method should involve the additional
assumption that the true mean response decreases generally with
increasing concentration. The researcher may also be interested in
determining significant differences between concentrations.
(4) Growth data should also be analyzed by one-way ANOVA with the
inclusion of a covariate to account for possible differences in growth
of surviving fry in embryo cup(s) that contain fewer individuals. This
condition can occur in cases when the same amount of food is given to
each test chamber regardless of the number of survivors.
(B) Test data to be analyzed. Data to be statistically analyzed are:
(1) Percentage of healthy, fertile embryos at 40-48 hours after
initiation of the test. Percentage is based upon initial number used.
(2) Percentage of embryos that produce live fry for release into
test chambers. Percentage is based on number of embryos remaining after
thinning.
(3) Percentage of embryos that produce live, normal fry for release
into test chambers. Percentage is based upon number of embryos remaining
after thinning.
(4) Percentage of fry survival at swim-up for trout. Percentage is
based upon number of embryos remaining after thinning.
(5) Percentage of embryos that produce live fish at end of test.
Percentage is based upon number of embryos remaining after thinning.
(6) Percentage of embryos that produce live, normal fish at end of
test. Percentage is based upon number of embryos remaining after
thinning.
(7) Weights and lengths of individual fish alive at the end of the
test.
(C) It is important that fish length and weight measurements be
associated with individual test chambers since the density of the fish
and available food should be considered in the growth of the organism.
(iii) Acceptability criteria. (A) An early life stage toxicity test
is not acceptable unless at least one of the following criteria is
significantly different (p=0.05) from control organisms when compared
with treated organisms, and the responses are concentration-dependent:
mortality of embryos, hatching success, mortality of fry (at swim-up for
trout), total mortality throughout the test, and growth (i.e. weight).
If no significant effects occur, but the concentrations tested were the
highest possible due to solubility or other physio-chemical limitations,
the data will be considered for acceptance.
(B) In addition to obtaining significant effects on the exposed test
species, a measure of acceptability in the response of control fish is
also required.
(C) A test is not acceptable if the average survival of the control
fish at the end of the test is less than 80 percent or if survival in
any one control chamber is less than 70 percent. For silversides, a test
is not acceptable if the average overall survival of the control embryos
and fish at the end of the test is less than 60 percent.
(D) If a carrier is used, the criteria for effect (mortality of
embryos and fry, growth, etc.) used in the comparison of control and
exposed test organisms shall also be applied to the control and control
with carrier chambers. For the test to be considered acceptable, no
significant difference shall exist between these criteria.
(E) A test is not acceptable if the relative standard deviation
(RSD=100 times the standard deviation divided by the mean) of the
weights of the fish that were alive at the end of the test in any
control test chamber is greater than 40 percent.
(6) Analytical measurements--(i) Analysis of water quality.
Measurement of certain dilution water quality parameters shall be
performed every 6 months, to determine the consistency of the dilution
water quality. In addition, if data in 30-day increments are not
available to show that freshwater dilution water is constant,
measurements of hardness, alkalinity, pH, acidity, conductivity, TOC or
COD and particulate matter should be conducted once a week in the
highest test substance concentration. Measurement of
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calcium, magnesium, sodium, potassium, chloride, and sulfate is
desirable.
(ii) Dissolved oxygen measurement. The dissolved oxygen
concentration shall be measured in each test chamber at the beginning of
the test and at least once weekly thereafter (as long as live organisms
are present) in two replicates of the control and the high, medium, and
low test substance concentrations.
(iii) Temperature measurement. Temperatures shall be recorded in all
test chambers at the beginning of the test, once weekly thereafter and
at least hourly in one test chamber. When possible, the hourly
measurement shall be alternated between test chambers and between
replicates.
(iv) Test substance measurement. (A) Prior to the addition of the
test substance to the dilution water, it is recommended that the test
substance stock solution be analyzed to verify the concentration. After
addition of the test substance, the concentration of test substance
should be measured at the beginning of the test in each test
concentration and control(s), and at least once a week thereafter. Equal
aliquots of test solution may be removed from each replicate chamber and
pooled for analysis. If a malfunction in the delivery system is
discovered, water samples shall be taken from the affected test chambers
immediately and analyzed.
(B) The measured concentration of test substance in any chamber
should be no more than 30 percent higher or lower than the concentration
calculated from the composition of the stock solution and the
calibration of the test substance delivery system. If the difference is
more than 30 percent, the concentration of test substance in the
solution flowing into the exposure chamber (influent) should be
analyzed. These results will indicate whether the problem is in the
stock solution, the test substance delivery system or in the test
chamber. Measurement of degradation products of the test substance is
recommended if a reduction of the test substance concentration occurs in
the test chamber.
(v) Sampling and analysis methodology. (A) Generally, total test
substance measurements are sufficient; however, the chemical
characteristics of the test substance may require both dissolved and
suspended test substance measurements.
(B) For measurement of the test substance, water samples shall be
taken midway between the top, bottom, and sides of the test chamber and
should not include any surface scum or material stirred up from the
bottom or sides. Samples of test solutions shall be handled and stored
appropriately to minimize loss of test substance by microbial
degradation, photodegradation, chemical reaction, volatilization, or
sorption.
(C) Chemical and physical analyses shall be performed using
standardized methods whenever possible. The analytical method used to
measure the concentration of the test substance in the test solution
shall be validated before the beginning of the test. At a minimum, a
measure of the accuracy of the method should be obtained on each of two
separate days by using the method of known additions, and using dilution
water from a tank containing test organisms. Three samples should be
analyzed at the next-to-lowest test substance concentration. It is also
desirable to study the accuracy and precision of the analytical method
for test guideline determination by use of reference (split) samples, or
interlaboratory studies, and by comparison with alternative, reference,
or corroborative methods of analysis.
(D) An analytical method is not acceptable if likely degradation
products of the test substance, such as hydrolysis and oxidation
products, give positive or negative interferences, unless it is shown
that such degradation products are not present in the test chambers
during the test. In general, atomic absorption spectrophotometric
methods for metals and gas chromatographic methods for organic compounds
are preferable to colorimetric methods.
(E) In addition to analyzing samples of test solution, at least one
reagent blank also should be analyzed when a reagent is used in the
analysis. Also, at least one sample for the method of known additions
should be prepared by adding test substance at the concentration used in
the toxicity test.
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(d) Test conditions--(1) Test species. (i) One or more of the
recommended test species will be specified in rules under part 799 of
this chapter requiring testing of specific chemicals. The recommended
test species are:
(A) Fathead minnow (Pimephales promelas Rafinesque).
(B) Sheepshead minnow (Cyprinodon variegatus).
(C) Brook trout (Salvelinus fontinalis).
(D) Rainbow trout (Salmo gairdneri).
(E) Atlantic silverside (Menidia menidia).
(F) Tidewater silverside (Menidia peninsulae).
(ii) Embryos used to initiate the early life stage test shall be
less than 48 hours old for the fathead and sheepshead minnows,
silversides, and less than 96 hours old for the brook trout and rainbow
trout. In addition, the following requirements shall be met:
(A) All embryos used in the test shall be from the same source.
Embryos shall be obtained from a stock cultured in-house when possible,
and maintained under the same parameters as specified for the test
conditions. When it is necessary to obtain embryos from an external
source, caution should be exercised to ensure embryo viability and to
minimize the possibility of fungal growth. A description of the brood
stock history or embryo source shall be made available to EPA upon
request.
(B) Test species shall be cared for and handled properly in order to
avoid unnecessary stress. To maintain test species in good condition and
to maximize growth, crowding shall be prevented, and the dissolved
oxygen level shall be maintained near saturation.
(C) Embryos and fish shall be handled as little as possible. Embryos
shall be counted and periodically inspected until hatching begins. When
larvae begin to hatch, they shall not be handled. Transfer of minnow
larvae from embryo cups to test chambers shall not involve the use of
nets. No handling is necessary following introduction into the test
chambers until termination of the test.
(D) If fathead minnow embryos are obtained from in-house culture
units, the embryos should be gently removed from the spawning substrate.
The method for separating the fertilized eggs from the substrate is
important and can affect the viability of the embryos; therefore the
finger-rolling procedure is recommended.
(E) Disease treatment. Chemical treatments to cure or prevent
diseases should not be used before, and should not be used during a
test. All prior treatments of brood stock should be reported in detail.
Severely diseased organisms should be destroyed.
(2) Test facilities--(i) Construction materials. Construction
materials and equipment that contact stock solutions, test solutions, or
dilution water into which test embryos or fish are placed should not
contain any substances that can be leached or dissolved into aqueous
solutions in quantities that can affect test results. Materials and
equipment that contact stock or test solutions should be chosen to
minimize sorption of test chemicals from dilution water. Glass,
316 stainless steel, nylon screen and perfluorocarbon plastic
(e.g., Teflon) are acceptable materials. Concrete or rigid
(unplasticized) plastic may be used for holding and acclimation tanks,
and for water supply systems, but they should be thoroughly conditioned
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. Natural rubber, copper, brass, galvanized
metal, epoxy glues, and flexible tubing should not come in contact with
dilution water, stock solutions, or test solutions.
(ii) Test chambers (exposure chambers). (A) Stainless steel test
chambers should be welded or glued with silicone adhesive, and not
soldered. Glass should be fused or bonded using clear silicone adhesive.
Epoxy glues are not recommended, but if used ample curing time should be
allowed prior to use. As little adhesive as possible should be in
contact with the water.
(B) Many different sizes of test chambers have been used
successfully. The size, shape and depth of the test chamber is
acceptable if the specified flow rate and loading requirements can be
achieved.
(C) The actual arrangement of the test chambers can be important to
the statistical analysis of the test data.
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Test chambers can be arranged totally on one level (tier) side by side,
or on two levels with each level having one of the replicate test
substance concentrations or controls. Regardless of the arrangement, it
shall be reported in detail and considered in the data analysis.
(iii) Embryo incubation apparatus. (A) Recommended embryo incubation
apparatus include embryo cups for the minnow species and screen trays
for the trout species, although embryo cups can be used for the trout
species. Embryo cups are normally constructed from approximately 4-5 cm
inside diameter, 7-8 cm high, glass jars with the end cut off or similar
sized sections of polyethylene tubing. One end of the jar or tubing is
covered with stainless steel or nylon screen (approximately 40 meshes
per inch is recommended). Embryo cups for silversides are normally
constructed by using silicone adhesive to glue a 10-cm high, 363-um
nylon mesh tube inside a 9-cm I.D. glass Petri dish bottom. The embryo
cups shall be appropriately labeled and then suspended in the test
chamber in such a manner as to ensure that the test solution regularly
flows through the cup and that the embryos are always submerged but are
not agitated too vigorously. Cups may be oscillated by a rocker arm
apparatus with a low rpm motor (e.g., 2 rpm) to maintain the required
flow of test water. The vertical-travel distance of the rocker arm
apparatus during oscillation is normally 2.5-4.0 cm. The water level in
the test chambers may also be varied by means of a self-starting siphon
in order to ensure exchange of water in the embryo cups.
(B) The trout embryo incubation trays can be made from stainless
steel screen (or other acceptable material such as plastic) of about 3-4
mm mesh. The screen tray should be supported above the bottom of the
test chamber by two folds of screen or other devices which function as
legs or supports. The edges of the screen tray should be turned up to
prevent bump spills and to prevent the embryos from rolling off in the
event of excessive turbulence. Suspending or supporting the screen tray
off the bottom ensures adequate water circulation around the embryos and
avoids contact of embryos with possible bottom debris.
(iv) Test substance delivery system. (A) The choice of a specific
delivery system depends upon the specific properties and requirements of
the test substance. The apparatus used should accurately and precisely
deliver the appropriate amount of stock solution and dilution water to
the test chambers. The system selected shall be calibrated before each
test. Calibration includes determining the flow rate through each
chamber, and the proportion of stock solution to dilution water
delivered to each chamber. The general operation of the test substance
delivery system shall be checked at least twice daily for normal
operation throughout the test. A minimum of five test substance
concentrations and one control shall be used for each test.
(B) The proportional diluter and modified proportional diluter
systems and metering pump systems have proven suitable and have received
extensive use.
(C) Mixing chambers shall be used between the diluter and the test
chamber(s). This may be a small container or flow-splitting chamber to
promote mixing of test substance stock solution and dilution water, and
is positioned between the diluter and the test chambers for each
concentration. If a proportional diluter is used, separate delivery
tubes shall run from the flow-splitting chamber to each replicate test
chamber. Daily checks on this latter system shall be made.
(D) Silverside fry are injured easily and are susceptible to
impingement on the mesh of the incubation cups. Consequently, water flow
into and out of the cups when counting fry must be at a slow rate. This
can be accomplished by using small diameter (e.g., 2 mm I.D.) capillary
tubes to drain the test solution from spitter boxes into the replicate
test chambers. The use of a self-starting siphon to gradually lower
(i.e., less than or equal to 1 min.) the water level approximately 2 cm
in the test chamber is recommended. A minimum water depth of 5 cm should
be maintained in the cups. Although it may be satisfactory, a rocker-arm
type apparatus has not yet been used with silversides.
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(v) Other equipment required. (A) An apparatus for removing
undesirable organisms, particulate matter and air bubbles.
(B) An apparatus for aerating water.
(C) A suitable magnifying viewer for examination of minnow embryos.
(D) A suitable apparatus for the precise measurement of growth of
the fish, including both length (e.g., with metric or ruler caliper or
photographic equipment) and weight.
(E) Facilities for providing a continuous supply of live brine
shrimp nauplii (Artemia salina).
(F) For silversides, facilities for providing a supply of rotifers
(Brachionus plicatilis) for approximately 11 days.
(G) Facilities (or access to facilities) for performing the required
water chemistry analyses.
(vi) Cleaning of equipment. (A) Test substance delivery systems and
test chambers should be cleaned before use. Test chambers should be
cleaned during the test as needed to maintain the dissolved oxygen
concentration, and to prevent clogging of the embryo cup screens and
narrow flow passages.
(B) Debris can be removed with a rubber bulb and large pipette or by
siphoning with a glass tube attached to a flexible hose. Debris should
be run into a bucket light enough to observe that no live fish are
accidentally discarded.
(vii) Dilution water--(A) General. (1) A constant supply of
acceptable dilution water should be available for use throughout the
test. Dilution water shall be of a minimum quality such that the test
species selected will survive in it for the duration of testing without
showing signs of stress (e.g., loss of pigmentation, disorientation,
poor response to external stimuli, excessive mucous secretion, lethargy,
lack of feeding, or other unusual behavior). A better criterion for an
acceptable dilution water for tests on early life stages should be such
that the species selected for testing will survive, grow, and reproduce
satisfactorily in it.
(2) The concentration of dissolved oxygen in the dilution water
(fresh or salt) shall be between 90 percent and 100 percent saturation.
When necessary, dilution water should be aerated by means of airstones,
surface aerators, or screen tubes before the introduction of the test
substance.
(3) Water that is contaminated with undesirable microoganisms (e.g.,
fish pathogens) shall not be used. If such contamination is suspected,
the water should be passed through a properly maintained ultraviolet
sterilizer equipped with an intensity meter before use. Efficacy of the
sterilizer can be determined by using standard plate count methods.
(B) Freshwater. (1) Natural water (clean surface or ground water) is
preferred, however, dechlorinated tap water may be used as a last
resort. Reconstituted freshwater is not recommended as a practical
dilution water for the early life stage toxicity test because of the
large volume of water required.
(2) Particulate and dissolved substance concentrations should be
measured at least twice a year and should meet the following
specifications:
------------------------------------------------------------------------
Substance Concentration maximum
------------------------------------------------------------------------
Particulate matter....................... <20 mg/liter.
Total organic carbon (TOC)............... <2 mg/liter.
Chemical oxygen demand (COD)............. <5 mg/liter.
Un-ionized ammonia....................... <1 [micro]g/liter.
Residual chlorine........................ <1 [micro]g/liter.
Total organoposphorus pesticides......... <50 ng/liter.
Total organochlorine pesticides plus <50 ng/liter.
polychlorinated biphenyls (PCBs).
Total organic chlorine................... <25 ng/liter.
------------------------------------------------------------------------
(3) During any one month, freshwater dilution water should not vary
more than 10 percent from the respective monthly averages of hardness,
alkalinity and specific conductance; the monthly pH range should be less
than 0.4 pH units.
(C) Saltwater. (1) Marine dilution water is considered to be of
constant quality if the minimum salinity is greater than 15\0\/
00 and the weekly range of the salinity is less than 15\0\/
00. The monthly range of pH shall be less than 0.8 pH units.
Saltwater shall be filtered to remove larval predators. A pore size of
<=20 micrometers ([micro]m) is recommended. For silversides, the
recommended salinity is 20 ppt and shall be maintained between 15 and 25
ppt throughout testing.
(2) Artificial sea salts may be added to natural seawater during
periods of
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low salinity to maintain salinity above 15\0\/00.
(3) Test parameters--(i) Dissolved oxygen concentration. It is
recommended that the dissolved oxygen concentration be maintained
between 90 and 100 percent saturation; but it shall be no less than 75
percent saturation at all times for both minnow species and between 90
and 100 percent saturation for the trout species in all test chambers.
Dilution water in the head box may be aerated, but the test solution
itself shall not be aerated.
(ii) Loading and flow rate. (A) The loading in test chambers should
not exceed 0.1 grams of fish per liter of test solution passing through
the test chamber in 24 hours. The flow rate to each chamber should be a
minimum of 6 tank volumes per 24 hours. During a test, the flow rates
should not vary more than 10 percent from any one test chamber to any
other.
(B) A lower loading or higher flow rate or both shall be used if
necessary to meet the following three criteria at all times during the
test in each chamber containing live test organisms:
(1) The concentration of dissolved oxygen shall not fall below 75
percent saturation for the fathead and sheepshead minnows and 90 percent
for the rainbow and brook trout;
(2) The concentration of un-ionized ammonia should not exceed 1
[micro]g/1; and
(3) The concentration of toxicant should not be lowered (i.e.,
caused by uptake by the test organisms and/or materials on the sides and
bottoms of the chambers) more than 20 percent of the mean measured
concentration.
(iii) Temperature. (A) The recommended test temperatures are:
(1) Fathead minnow--25 [deg]C for all life stages.
(2) Sheepshead minnow--30 [deg]C for all life stages.
(3) Rainbow and brook trout--10 [deg]C for embryos. 12 [deg]C for
fry and alevins.
(4) Atlantic and tidewater silversides--25 [deg]C for all life
stages.
(B) Excursions from the test temperature shall be no greater than
2.0[deg]C. It is recommended that the test system
be equipped with an automatic alarm system to alert staff of
instantaneous temperature changes in excess of 2 [deg]C. If the water is
heated (i.e., for minnow species), precautions should be taken to ensure
that supersaturation of dissolved gases is avoided. Temperatures shall
be recorded in all test chambers at the beginning of the test and weekly
thereafter. The temperature shall be recorded at least hourly in one
test chamber throughout the test.
(iv) Light. (A) Brook and rainbow trout embryos shall be maintained
in darkness or very low light intensity through one week post-hatch, at
which time a 14-hour light and 10-hour dark photoperiod shall be
provided.
(B) For fathead and sheepshead minnows, a 16-hour light and 8-hour
dark (or 12:12) photoperiod shall be used throughout the test period.
(C) For silversides, a 14-hour light and 10-hour dark photoperiod
shall be used throughout the test period.
(D) A 15-minute to 30-minute transition period between light and
dark is optional.
(E) Light intensities ranging from 30 to 100 lumens at the water
surface shall be provided; the intensity selected should be duplicated
as closely as possible for all test chambers.
(e) Reporting. A report of the results of an early life stage
toxicity test shall include the following:
(1) Name of test, sponsor, investigator, laboratory, and dates of
test duration.
(2) Detailed description of the test substance including its source,
lot number, composition (identity and concentration of major ingredients
and major impurities), known physical and chemical properties, and any
carriers (solvents) or other additives used.
(3) The source of the dilution water, its chemical characteristics,
and a description of any pretreatment.
(4) Detailed information about the test organisms including
scientific name and how verified and source history, observed diseases,
treatments, acclimation procedure, and concentration of any contaminants
and the method of measurement.
(5) A description of the experimental design and the test chambers,
the depth and volume of the solution in the chambers, the way the test
was begun, the number of organisms per treatment, the number of
replicates, the
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loading, the lighting, a description of the test substance delivery
system, and the flow rate as volume additions per 24 hours.
(6) Detailed information on feeding of fish during the toxicity
test, including type of food used, its source, feeding frequency and
results of analysis (i.e., concentrations) for contaminants.
(7) Number of embryos hatched, number of healthy embryos, time to
hatch, mortality of embryos and fry, measurements of growth (weight and
length), incidence of pathological or histological effects and
observations of other effects or clinical signs, number of healthy fish
at end of test.
(8) Number of organisms that died or showed an effect in the control
and the results of analysis for concentration(s) of any contaminant in
the control(s) should mortality occur.
(9) Methods used for, and the results of (with standard deviation),
all chemical analyses of water quality and test substance concentration,
including validation studies and reagent blanks; the average and range
of the test temperature(s).
(10) Anything unusual about the test, any deviation from these
procedures, and any other relevant information.
(11) A description of any abnormal effects and the number of fish
which were affected during each period between observations in each
chamber, and the average concentration of test substance in each test
chamber.
(12) Reference to the raw data location.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19064, May 20, 1987]