United States      Prevention, Pesticides     EPA 712-C-96-117
          Environmental Protection    and Toxic Substances     April 1996
          Agency        (7101)
&EPA   Ecological Effects Test
          OPPTS 850.1085
          Fish Acute Toxicity
          Mitigated by Humic Acid
                'Public Draft"

     This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.

     The Office of Prevention,  Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a  process of harmonization that
blended the testing  guidance and requirements that existed in the Office
of Pollution Prevention and Toxics  (OPPT) and appeared in Title 40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development

     The purpose of harmonizing these guidelines into a single set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic  Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide,  Fungicide and Rodenticide Act
(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:

     To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency,  401  M  St.  SW.,  Washington, DC 20460. In  person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted  electronically by  sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.

     Final  Guideline Release: This guideline is available  from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin  Board.   By  modem   dial   202-512-1387,   telnet   and  ftp:
fedbbs.access.gpo.gov (IP,  or  call 202-512-0135 for disks
or paper copies.  This  guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access  Gopher
(gopher.epa.gov) under the heading  "Environmental Test Methods and

OPPTS 850.1085   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

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

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-

     (v) The mortality data are  used to calculate LC50  values and their
95 percent confidence limits, and to plot concentration-response  curves

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

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-

     (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

      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
      Maximum Concentration
Total suspended solids  	
Total organic  carbon  (TOC), or chemical  oxygen  demand
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.

     (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-

     (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-

     (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).

     (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

     (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.