United States Prevention, Pesticides EPA 712-C-96-117
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1085
Fish Acute Toxicity
Mitigated by Humic Acid
'Public Draft"
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INTRODUCTION
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
(OECD).
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
(7U.S.C. I36,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:
guidelines@epamail.epa.gov.
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 162.140.64.19), 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
Guidelines."
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OPPTS 850.1085 Fish acute toxicity mitigated by humic acid.
(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 797.1460 Fish Acute Toxicity
Mitigated by Humic Acid.
(b) Purpose. This guideline may be used to develop data on the acute
toxicity of chemical substances and mixtures under static or static renewal
conditions, subject to environmental effects testing. This guideline pre-
scribes procedures to be used to develop data on the acute toxicity of
chemicals to fish with and without the presence of naturally occurring dis-
solved organic substances (.e.g., humic acids and their salts). EPA will
use data from these tests in assessing the hazard of a chemical to the envi-
ronment. For additional background information on this test guideline see
OPPTS 850.1075.
(c) Definitions. In addition to the definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in 40 CFR Part 792—
Good Laboratory Practice Standards, the following definitions also apply
to this test guideline:
Acclimation means the physiological compensation by test organisms
to new environmental conditions (e.g. temperature, hardness, pH).
Acute toxicity test means a method used to determine the concentra-
tion of a substance that produces a toxic effect on a specified percentage
of test organisms in a short period of time (e.g. 96 h). In this guideline,
death is used as the measure of toxicity under static or static renewal con-
ditions only.
Carrier means a solvent used to dissolve a test substance prior to
delivery to the test chamber.
Death means the lack of opercular movement by a test fish.
Dissolved organic carbon (DOC) means various organic molecules
occurring in lotic and lentic ecosystems, which in this test are restricted
to a heterogeneous group of humic substances.
Total organic carbon (TOC) means the sum of all organic carbon
molecules, which are dissolved, particulate, and suspended, occurring in
test dilution waters.
Humic substances means humic acids (HAs), fulvic acids, and humin
fractions, and their various salts, resulting from chemical fractionation of
this heterogeneous naturally-occurring organic substance. For purposes of
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this test, HA, sodium salt (e.g. Aldrich Catalog No. HI,675-2; mention
of a commercial company or product does not constitute approval or en-
dorsement by the Agency) may be used as the source of DOC.
LC50 means that the test substance concentration calculated from ex-
perimentally-derived mortality data is lethal to 50 percent of a test popu-
lation during continuous exposure over a specified period of time.
Loading means the ratio of fish 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.
Static means the test solution is not renewed during the period of
the test.
Test solution means the dilution water containing the dissolved test
substance to which test organisms are exposed.
(d) Test procedures—(1) Summary of the test, (i) This test is de-
signed to determine the acute effects of the test substance on one of three
species of fish with HA. Test chambers are filled with appropriate volumes
of dilution water.
(ii) The test substance is introduced into each test chamber. Some
test chambers contain only dilution water; other contain a concentration
of spiked HA.
(iii) Test fish which have been acclimated in accordance with the
test design are introduced into the test and control chambers by stratified
random assignment.
(iv) Fish in the test and control chambers are observed periodically
during the test; dead fish are removed at least twice each day and the
findings are recorded.
(v) The dissolved oxygen (DO) concentration, pH, and temperature
are measured at intervals in selected test chambers.
(vi) A concentration-response curve and LC50 value for the test sub-
stance in dilution water spiked with a known amount of HA are developed
from the mortality data collected during the test.
(2) Range-finding test, (i) If the toxicity of the test substance in HA
is not already known, a range-finding test should be performed to deter-
mine the range of concentrations to be used in the definitive test. The
highest concentration of test substance for use in the range-finding test
should not exceed its solubility in water or the permissible amount of car-
rier used.
(ii) Initially, two fish test is performed at 20 mg/L of HA. In some
cases, the 20 mg HA/L concentration may be so high that no toxicity will
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be present due to the formation of viscous, colloidal complexes. If this
occurs, the 20 mg HA/L concentration should be decreased to 15 mg/L,
or an appropriately lower concentration.
(3) Definitive test, (i) A minimum of 20 fish should be exposed to
each of five or more test substance concentrations in dilution water spiked
with a known amount of HA. The range of test substance concentrations
to which the fish are exposed should be such that in 96 h there are at
least two partial mortality exposures bracketing 50 percent survival.
(ii) For exposure to each concentration of a test substance in dilution
water spiked with a known amount of HA, an equal number of test fish
should be placed in two or more replicate test chambers. Test fish should
be impartially distributed among test chambers in such a manner that test
results show no significant bias from the distributions.
(iii) Every test should include a control consisting of the same dilution
water, conditions, procedures, and fish from the same group used in the
test, except that none of the test substance is added. Every test should
also include negative controls consisting of dilution water with HA alone.
(iv) Mortality data collected during the test are used to calculate a
96-h LC50 value. The 24-, 48-, and 72-h values should be calculated
whenever there is sufficient mortality data to determine such values.
(v) Test fish should not be fed while they are being exposed to the
test substance under static conditions.
(4) Test results, (i) Death is the primary criterion used in this test
guideline to evaluate the toxicity of the test substances on the presence
of a known amount of HA.
(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 of physiology, such as discoloration, exces-
sive mucous production, hyperventilation, opaque eyes, curved spine, or
hemorrhaging should be recorded.
(iii) Observations on compound solubility and/or dispersibility should
be recorded. The investigator should report the appearance of surface
slicks, precipitates, or material adhering to the sides of the test chamber.
(iv) Each test and control chamber should be checked for dead fish
and observations recorded at 24, 48, 72, and 96 h after the beginning of
the test or within 1 h of the designated times. If the test is continued
past 96 h, additional observations should be made every 24 h until termi-
nation.
(v) The mortality data are used to calculate LC50 values and their
95 percent confidence limits, and to plot concentration-response curves
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for each time interval whenever sufficient data exists. The methods rec-
ommended for use in calculating LC50 values include probit, logit, bino-
mial, and moving average angle.
(vi) A test is be unacceptable if more than 10 percent of the control
fish die or exhibit abnormal behavior during a 96-h test.
(5) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, TOC, or chemical oxygen
demand (COD), and total suspended solids (TSS) of the dilution water
should be measured at the beginning of each static 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 and end of
the test. The test solution volume should not be reduced by more than
10 percent as a result of these measurements.
(ii) Dissolved organic carbon. The naturally-occurring DOC selected
for this test should be HA, which is available from the Aldrich catalog,
(No. Hl,675-2).
(iii) Collection of samples for measurement of TOC. Samples to
be analyzed for TOC should be taken from the control chambers midway
between the top, bottom, and sides of the test chamber. These samples
should not include any surface scum or material dislodged from the bottom
or sides.
(iv) Measurement of TOC. (A) For static tests, DOC should be
measured (as TOC) at a minimum in each test chamber at the beginning
(time 0, before fish are added) of the test. Three TOC measurements
should be made and the average reported.
(B) The analytical methods used to measure the TOC in a sample
should be validated before beginning the test. The accuracy of a method
should be verified by a method such as using known additions. This in-
volves adding a known amount of the dissolved organic carbon source
to three water samples taken from a chamber containing dilution water
to be used in the test. The normal concentration of dissolved organic car-
bon in those samples should span the TOC concentration range to be used
in the test.
(C) The nominal concentration of test substance based on 100 percent
active ingredient (AI) should be used to calculate all LC50 values and
to plot all concentration-response curves.
(e) Test conditions—(1) Test species— (i) Selection. The test spe-
cies for this test are the rainbow trout (Oncorhynchus mykiss = Salmo
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gairdnerf), bluegill (Lepomis macrochirus), and fathead minnow
(Pimephales promelas).
(ii) Age and condition of fish. Juvenile fish should be used. Fish
used in a particular test should be the same age and be of normal size
and appearance for their age. The longest fish should not be more than
twice the length of the shortest. All newly acquired fish should be quar-
antine and observed for at least 14 days prior to use in a test. Fish 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 test.
(iii) Acclimation of test fish. 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. The fish should be held
an additional 14 days in the dilution water prior to testing. Any changes
in water temperature should not exceed 3 °C per day. Fish should be held
for a minimum of 7 days at the test temperature prior to testing. During
the final 48-h of acclimation, fish should be maintained in facilities with
background colors and light intensities similar to those of the testing area
and should not be fed.
(2) Facilities—(i) General. Facilities needed to perform this test in-
clude:
(A) Flow-through tanks for holding and acclimating fish.
(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 insufficient dissolved oxygen, respectively.
(D) Apparatus for providing a 16-h light and 8-h dark photoperiod
with a 15- to 30-min transition period.
(E) Chambers for exposing test fish to the test substance.
(ii) Construction materials. Construction materials and commer-
cially purchased equipment that may contact the stock solution, test solu-
tion, or dilution water should not contain substances that can be leached
or dissolved into aqueous solutions in quantities that can alter the test re-
sults. Materials and equipment that contact stock or test solutions should
be chosen to minimize sorption of test chemicals. Glass, stainless steel,
and perfluorocarbon plastic should be used whenever possible. Concrete,
fiberglass, 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 or filters
should be used to remove rust particles. Rubber, copper, brass, galvanized
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metal, epoxy glues, and lead should not come in contact with the dilution
water, stock solution, or test solution.
(iii) 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.
(iv) Cleaning of test system. 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.
(v) Dilution water. (A) Clean surface or ground water, reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
the test fish will survive in it for the duration of the holding, acclimating,
and testing periods without showing signs of stress, such as discoloration,
hemorrhaging, disorientation, or other unusual behavior. The quality of
the clean dilution water (without spiked HA) should be constant and
should meet the specifications in the following Table 1., measured at least
twice a year:
Table 1.—Specifications for Dilution Water
Substance
Maximum Concentration
Total suspended solids
Total organic carbon (TOC), or chemical oxygen demand
(COD).
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls
(PCBs),or organic chlorine.
Hardness (as CaCO3 during testing)
20 mg/L
2 mg/L, or 5 mg/L, respectively
20 ug/L
1 ug/L
50 ng/L
50 ng/L, or 25 ng/L, respectively
180 mg/L
The quality of the dilution water after spiking with HA should meet all
the previous specifications except for TOC or COD.
(B) The DO concentration in the dilution water should be between
90 and 100 percent saturation; 9.8 to 10.9 mg/L for tests with trout, and
8.0 to 8.9 mg/L for tests with bluegill or fathead minnow 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.
Buffered soft water should be aerated before but not after the addition
of buffers.
(C) Diseased organisms present in the dilution water in sufficient
number to cause infection of the fish should be killed or removed by suit-
able equipment.
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(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 should be measured on each batch.
(vi) Carriers. Only distilled water should be used in making stock
solutions of the test substance. However, if the stock volume is more than
10 percent of the test solution volume, dilution water should be used. Car-
bon-based carriers cannot be used in this test. If necessary, stock solution
pH should be adjusted to pH 7.
(3) Test parameters—(i) Loading. The number of fish 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 concentrations
are decreased by more than 20 percent due to uptake by the fish. Loading
should not exceed 0.5 g of fish/L of solution in the test chamber at any
one time. These loading rates should be sufficient to maintain the DO
concentration above the recommended levels and the ammonia concentra-
tion below 20 (ig/L.
(ii) Dissolved oxygen concentration. During static tests with rainbow
trout, the DO should be maintained above 5.5 mg/L in each test chamber.
In tests with bluegill and fathead minnow, the DO should be greater than
4.5 mg/L in each test chamber.
(iii) Temperature. The test temperature should be 22 °C for bluegill
and fathead minnow, and 12 °C for rainbow trout. Deviations from the
test temperature should be no greater than ±2 °C. The temperature should
be measured at least hourly in one test chamber.
(iv) Light. A 16-h light and 8-h dark photoperiod should be main-
tained.
(f) Reporting. The sponsor should submit to the EPA all data devel-
oped by the test that are suggestive or predictive of toxicity. In addition
to the reporting requirements prescribed in 40 CFR Part 792—Good Lab-
oratory Practice Standards, the reported test data should include the follow-
ing:
(1) The source of the dilution water, a description of any pretreatment,
and the measured hardness, acidity, alkalinity, pH, conductivity, TOC,
COD, and total suspended solids.
(2) The source of the HA (e.g., batch number), as well as a complete
description and chemical characterization.
(3) A description of the test chambers, the depth and volume of solu-
tion in the chamber, and the specific way the test was begun (e.g., condi-
tioning and test substance additions).
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(4) Detailed information about the test fish, including the scientific
name and method of verification, average weight (grams, wet weight),
standard length, age, source, history, observed diseases, treatments and
mortalities, acclimation procedures, and food use.
(5) The number of replicates used, the number of organisms per rep-
licate, and the loading rate.
(6) The measured DO, pH, and temperature and the lighting regime.
(7) A description of preparation of the stock solution. If the pH of
the stock solution was adjusted, describe the adjustment.
(8) The concentrations of the dissolved organic carbon as TOC from
the HA control just before the start of the test, all triplicate measurements,
and average TOC values.
(9) Results from any range-finding tests performed at 20 mg/L of
HA.
(10) The number of dead and live tests organisms, the percentage
of organisms that died, and the number that showed any abnormal effects
in the control and in each test chamber at each observation period.
(11) The 96-h LC50, and when sufficient data have been generated,
the 24-, 48-, 72-h LC50 values, their 95 percent confidence limits, and
the methods used to calculate the LC50 values and their confidence limits.
(12) When observed, the no-observed-effect-concentration (the high-
est concentration tested at which there were no mortalities, abnormal be-
havioral, or physiological effects) in treatments.
(13) The concentration-response curve at each observation period for
which LC50 values are calculated.
(14) Methods and data records of all chemical analyses of water qual-
ity parameters, TOC, including method validations and reagent blanks.
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