United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–117

April 1996

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 published 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. Environmental 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, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized 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 prescribes procedures to be used to develop data on the acute toxicity of

chemicals to fish with and without the presence of naturally occurring dissolved 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 environment. 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 concentration 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 conditions 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. H1,675–2; mention

of a commercial company or product does not constitute approval or endorsement by the Agency) may be used as the source of DOC.

LC50 means that the test substance concentration calculated from experimentally-derived mortality data is lethal to 50 percent of a test population 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 designed 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 substance 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 determine 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 carrier 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 limited to, erratic swimming, loss of reflex, increased excitability, lethargy,

or any changes in appearance of physiology, such as discoloration, excessive 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 termination.

(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 recommended for use in calculating LC50 values include probit, logit, binomial, 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-tomonth 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. H1,675–2).

(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 involves 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 carbon 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 species for this test are the rainbow trout (Oncorhynchus mykiss = Salmo
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gairdneri), 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 quarantine 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 include:

(A) Flow-through tanks for holding and acclimating fish.

(B) A mechanism for controlling and maintaining the water temperature 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 commercially purchased equipment that may contact the stock solution, test 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 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 conditioned 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 ................................................................ 20 mg/L

Total organic carbon (TOC), or chemical oxygen demand

(COD).

2 mg/L, or 5 mg/L, respectively

Un-ionized ammonia .................................................................... 20 μg/L

Residual chlorine ......................................................................... 1 μg/L

Total organophosphorus pesticides ............................................. 50 ng/L

Total organochlorine pesticides plus polychlorinated biphenyls

(PCBs),or organic chlorine.

50 ng/L, or 25 ng/L, respectively

Hardness (as CaCO3 during testing) ........................................... 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 suitable equipment.
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(D) Glass-distilled or carbon-filtered deionized water with a conductivity less than 1 μS/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. Carbon-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 concentration below 20 μg/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 maintained.

(f) Reporting. The sponsor should submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In addition

to the reporting requirements prescribed in 40 CFR Part 792—Good Laboratory Practice Standards, the reported test data should include the following:

(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 solution in the chamber, and the specific way the test was begun (e.g., conditioning 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 replicate, 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 highest concentration tested at which there were no mortalities, abnormal behavioral, 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 quality parameters, TOC, including method validations and reagent blanks.