United States       Prevention, Pesticides     EPA712-C-96-130
          Environmental Protection    and Toxic Substances     April 1996
          Agency        (7101)
&EPA   Ecological Effects Test
          OPPTS 850.1020
          Gammarid Acute
          Toxicity Test
                'Public Draft"

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

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

     The purpose of harmonizing these guidelines into a single set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic  Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide,  Fungicide and Rodenticide Act
(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 850.1020   Gammarid acute toxicity test
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing  requirements   of both  the  Federal  Insecticide,  Fungicide,  and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

     (2) Background. The source material  used in developing this har-
monized OPPTS test guideline is 40 CFR 795.120 Gammarid Acute Tox-
icity Test.

     (a) Purpose.  This guideline is  intended for use in developing data
on the acute toxicity of chemical substances and mixtures subject to envi-
ronmental effects test regulations. This guideline describes a test  to de-
velop data on the acute toxicity of chemicals to  gammarids. The data from
this  test will be used in  assessing the hazard of a chemical  to aquatic

     (b) Definitions.  The definitions in section 3 of TSCA and in Part
792, Good Laboratory Practice Standards, apply to this test guideline. The
following definitions also apply to this guideline:

     Death means  the lack of reaction of a  test organism to gentle prod-

     Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or acclima-
tion tank, with no recycling.

     LC50 means the median lethal concentration, i.e., that concentration
of a chemical in air or water killing 50 percent of the test batch of orga-
nisms within a particular period of exposure (which shall be stated).

     Loading means the ratio  of the biomass of gammarids (grams,  wet
weight) to the volume (liters)  of test solution in either a test chamber or
passing through it in a 24-hour period.

     Solvent means a  substance  (e.g., acetone) which is combined with
the test substance  to  facilitate introduction of the test substance  into the
dilution water.

     Static system  means a test chamber in which the  test solution is not
renewed during the period of the test.

     (c) Test procedures—(1) Summary of the test. In preparation for
the test, test chambers are filled with  appropriate volumes  of dilution
water.  If a  flow-through  test is performed, the flow  of dilution water
through each chamber is adjusted to the rate desired. In a static test, the
test substance is introduced into  each test chamber. In a flow-through test,
the rate in which the test substance  is added is adjusted to establish  and
maintain the desired concentration of test substance in each test chamber.

The test is started by randomly introducing gammarids, which have been
acclimated to the  test conditions, into the test  chambers. Gammarids in
the test chambers  are  observed periodically during  the test;  the dead
gammarids are removed and the findings recorded. Dissolved oxygen con-
centration, pH, temperature, and the concentration of test substance in test
chambers  are measured at specified intervals. Data collected during the
test are used to develop concentration—response curves and LC50 values
for the test substance.

    (2) Range-finding test, (i) A range-finding test should be conducted
to establish test substance concentrations to be used for the definitive test.

    (ii) The gammarids shall be exposed to a wide-range of concentrations
of the test substance (e.g. 1,  10, 100 mg/L, etc.), usually under static condi-

    (iii) A minimum of five gammarids should be exposed to each con-
centration of test substance for a period of 96 hours. The  exposure period
may be shortened if data suitable for determining concentrations in the
definitive  test can be obtained in less time. Nominal concentrations of the
test substance may be acceptable.

    (3) Definitive test, (i)  The purpose of the definitive test is to deter-
mine  the  24, 48, 72, and 96—hour LC50 values and the concentration-
response curves.

    (ii) A minimum of 20 gammarids per concentration shall be exposed
to five or  more concentrations of the test substance chosen in a geometric
series in which  the ratio is between 1.5 and 2.0 (e.g.,  2, 4,  8,  16, 32,
64 mg/L). The range and number of concentrations to which the organisms
are exposed shall be such that in 96  hours there is at least one concentra-
tion resulting in mortality greater than 50 and less than  100 percent, and
one concentration causing greater than zero and  less than  50 percent mor-
tality. An  equal number of gammarids may be placed in two or more rep-
licate  test chambers. Solvents should be avoided, if possible. If solvents
have to be used,  a solvent control, as well as a dilution control, shall be
tested at the highest solvent concentration employed in  the treatments. The
solvent should not be  toxic or have an effect on the  toxicity of  the test
substance. The concentration of solvent should not exceed 0.1 ml/L.

    (iii) Every test shall include a  concurrent  control using gammarids
from  the  same population or culture container.  The control group shall
be exposed to the same dilution water, conditions and procedures, except
that none of the test substance shall be is added to the chamber.

    (iv) The dissolved oxygen  concentration, temperature and pH of the
test solution  shall be measured at the beginning of the test and at 24,
48, 72 and 96 hours in at least  one replicate each of the  control,  and the
highest, lowest and middle test concentrations.

     (v) The test duration is 96 hours.  The test is unacceptable if more
than 10 percent of the control organisms  die during the test.

     (vi) In  addition to death, any abnormal behavior or appearance shall
also be reported.

     (vii) Gammarids shall be randomly assigned to the test chambers. Test
chambers shall be positioned within the testing area in  a random manner
or in a way  in which appropriate statistical analyses can be used to deter-
mine whether there is any variation due to placement.

     (viii) Gammarids shall be introduced into the  test chambers after the
test substance has been added.

     (ix) Observations on compound solubility shall be  recorded. The in-
vestigator should record the appearance of surface  slicks, precipitates, or
material adhering to the sides of the test chambers.

     (4)  Analytical  measurements—(i)  Water quality  analysis.  The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particu-
late matter  of the dilution water shall be measured  at the beginning of
each definitive test.

     (ii) Collection of samples for measurement of test substance. Each
sample to be analyzed for the test substance concentrations shall be taken
at a location midway between the top, bottom, and sides of the test cham-
ber.  Samples should not include any surface scum or material dislodged
from the bottom or sides. Samples shall be analyzed immediately or han-
dled and stored  in  a manner which minimizes  loss  of  test  substance
through microbial degradation,  photogradation,  chemical reaction, vola-
tilization, or sorption.

     (iii) Measurement of test  substance. (A) For static  tests, the con-
centration of dissolved test  substance (that which passes  through a  0.45
micron filter) shall be measured  in each  test chamber at  least at the begin-
ning (0-hour, before gammarids are added) and at the end of the  test.
During flow-through tests, the concentration of dissolved test substance
shall be  measured in each test  chamber at least at 0 and 96-hours  and
in at least one chamber whenever a malfunction of the  test substance deliv-
ery system is observed.

     (B)  The analytical methods used to measure the  amount of test sub-
stance  in a  sample shall be validated before beginning the test. This in-
volves adding a known amount of the test  substance to each of three water
samples taken  from a chamber containing dilution water and the same
number of gammarids as  are placed in each test  chamber. The nominal
concentrations of the test substance in these samples should span the con-
centration range to be used in the test. Validation of the  analytical method

should be performed on at least two  separate days prior to starting  the

     (C) An analytical method is not acceptable if likely degradation prod-
ucts of the test substance  give positive or negative interferences, unless
it is shown that such degradation products are not present in the test cham-
bers during the test.

     (D) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent.  The measured concentration of the test
substance in  any chamber during the  test shall not vary more  than plus
or minus 30  percent from  the measured concentration in that chamber at
zero time.

     (E)  The  mean  measured concentration of  dissolved test  substance
shall be used to calculate all LCSO's and to plot all concentration-response

     (d) Test conditions for definitive test—(1) Test species—(i) Selec-
tion. (A)  The amphipods,  Gammarus fasciatus,  G. pseudolimnaeus,  and
G. lacustris are specified for this test.

     (B)  Gammarids can be  cultured in the laboratory or collected from
natural sources. If collected, they must be held in  the  laboratory for at
least 14 days prior to testing.

     (C) Gammarids used in a particular test shall be  of similar age and/
or size and from the same source or culture population.

     (ii) Acclimation. If the holding  water is from  the same  source  as
the dilution water, acclimation to the dilution water shall be done gradually
over a 48-hour period.  The gammarids then shall be held at least 7 days
in the  dilution water prior to testing.  Any  changes  in water temperature
should not exceed 2 °C per day. Gammarids should be held for a minimum
of 7 days at the test temperature prior to testing.

     (iii) Care and  handling. Gammarids shall be  cultured in  dilution
water under  similar environmental conditions  to those  used in the test.
Organisms shall be handled  as little  as possible. When handling is nec-
essary  it should be done as gently, carefully and quickly as possible. Dur-
ing culturing and acclimation, gammarids shall be observed carefully for
signs of stress and mortality. Dead and abnormal individuals shall be dis-

     (iv) Feeding. The  organisms shall not be  fed during  testing. During
culturing, holding, and  acclimation,  a  sufficient quantity of deciduous
leaves, such  as  maple,  aspen, or birch, should be placed in the culture
and holding  containers to cover the bottom  with several layers. These
leaves  should be aged for at least 30 days in a flow-through system before

putting them in aquaria. As these leaves are eaten, more aged leaves should
be added. Pelleted fish food may also be added.

     (2) Facilities—(i) Apparatus—(A) Facilities needed to perform this
test include:

     (7) Containers for culturing, acclimating and testing gammarids;

     (2) Containers for aging leaves under flow-through conditions;

     (3) A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation and test periods;

     (4) Apparatus for straining particulate matter, removing gas bubbles,
or aerating the dilution water, as necessary; and

     (5) An apparatus for providing a 16-h light and 8-h dark photoperiod
with a 15- to 30-minute transition period.

     (B) Facilities should be well ventilated and free of fumes and disturb-
ances that may affect the test organism.

     (C)  Test chambers shall be covered loosely to reduce the loss of test
solution  or dilution water  due to evaporation and to minimize the entry
of dust or other particulates into the solutions.

     (ii)  Construction  materials. Construction materials and equipment
that may contact the stock solution, test solution or dilution water should
not contain substances that can be leached or  dissolved into aqueous solu-
tions  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 substances. Glass, stainless steel, and perfluorocarbon plastic should
be used wherever possible. Concrete, fiberglass, or plastic (e.g., PVC) may
be used for holding tanks, acclimation tanks, and water supply systems,
but they  should be aged prior  to use.  Rubber, coopper,  brass, galvanized
metal, and lead  should not come in contact with the  dilution water, stock
solution,  or test solution.

     (iii) Test substance delivery system. In flow-through tests, diluters,
metering pump  systems or other  suitable devices shall be used to  deliver
the test substance to the test chambers. The system used shall be calibrated
before each test. The  general operation of the test substance delivery sys-
tem  shall be checked  twice daily during a test. The  24-h flow shall  be
equal to  at least five times the volume of the test chamber. During a test,
the flow rates  should not vary more than 10 percent from one test chamber
to another.

     (iv)  Test chambers.  Test chambers shall contain  at least one liter
of test solution.  Test  chambers made of stainless steel should be welded,

       not soldered.  Test chambers made of glass  should be glued using clear
       silicone adhesive. As little  adhesive  as possible  should be  left exposed
       in the interior of the chamber. A substrate, such as a bent piece of stainless
       steel screen, should be placed on the bottom of each test chamber to pro-
       vide cover for the gammarids.

           (v) Cleaning of test system. Test substance delivery systems and test
       chambers  should be cleaned before each test. They should be  washed with
       detergent  and then rinsed sequentially with clean water, pesticide-free ace-
       tone,  clean  water, and  5 percent nitric  acid, followed by two  or more
       changes of dilution water.

           (vi) Dilution water. (A) Clean surface or ground water,  reconstituted
       water, or dechlorinated tap water  is  acceptable  as dilution water  if
       gammarids will survive  in it  for the duration of the culturing, acclimating,
       and testing  periods without  showing signs  of strees. The  quality  of the
       dilution water should be constant  enough that the month-to-month vari-
       ation  in hardness, acidity, alkalinity,  conductivity, TOC or COD, and par-
       ticulate matter is  not more  than 10  percent. The pH should be constant
       within 0.4 unit. In addition, the dilution water should meet the  following
       specifications measured  at least twice a year:
Particulate matter
Total organic carbon (TOC) or
 chemical oxygen demand (COD) [[[
Boron, fluoride [[[
Un-ionized ammonia [[[
Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, zinc
Residual chlorine [[[
Cadmium, mercury, silver
Total organophosphorus pesticides
Total organochlorine pesticides plus:
 polychlorinated biphenyls (PCBs) or
 organic chlorine
20 mg/L
2 mg/L
5 mg/L
100 |ig/L
1 ng/L
1 H9/L.
100 ng/L
50 ng/L

50 ng/L
25 ng/L
           (B)  If the dilution water is from  a ground  or  surface water source,
       conductivity  and total organic carbon (TOC) or chemical oxygen demand
       (COD) shall be  measured.  Reconstituted water  can be made  by adding
       specific  amounts of  reagent-grade  chemicals to  deionized or  distilled
       water. Glass-distilled or carbon-filtered deionized water with  a  conductiv-
       ity less than  1  (imho/cm is  acceptable as the diluent for making reconsti-
       tuted water.

           (C)  The concentration of dissolved oxygen in the dilution water shall

     (ii) Dissolved oxygen concentration between 60 and 105 percent satu-

     (iii) The  number of gammarids placed in a test chamber shall not
be so great as to  affect the  results  of the test. Ten  gammarids per liter
is the recommended level of loading for the static test. Loading require-
ments for the  flow-through test will vary depending  on the flow rate of
dilution water. The loading  should not cause the dissolved oxygen con-
centration to fall below the recommended levels.

     (iv) Photoperiod of 16 hours light and 8 hours darkness.

     (e) Reporting. The sponsor shall submit to the  EPA all data devel-
oped by the test that  are suggestive  or predictive of toxicity.  In addition,
the test report  shall include, but not necessarily be limited to, the following

     (1) Name and address of the facility performing  the study and the
dates on which the study was  initiated and completed.

     (2) Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.

     (3) Statistical methods employed for analyzing the data.

     (4) The test substance identified by name,  Chemical Abstracts (CAS)
number or code number, source, lot or batch number,  strength, purity, and
composition, or other appropriate characteristics.

     (5) Stability of the test substance under the conditions  of the test.

     (6) A description of the methods used, including:

     (i)  The source of the dilution water, its chemical characteristics (e.g.,
hardness, pH, etc.) and a description of any pretreatment.

     (ii) A description of the test substance delivery system, test chambers,
the  depth and volume of solution in the chamber, the way the test was
begun (e.g., test substance addition),  the loading, the lighting, and the flow

     (iii) Frequency and methods of measurements and observations.

     (7) The scientific name, weight,  length, source, and history of the
organisms used, and the acclimation procedures  and food used.

     (8) The concentrations tested, the number of gammarids and replicates
per test concentration. The reported results should include:

     (i)  The results of dissolved  oxygen, pH  and temperature measure-

     (ii) If solvents are used, the name and source of the solvent, the nomi-
nal concentration of the test substance in the  stock solution, the highest
solvent concentration in the test solution and a description of the solubility
determination in water and solvents.

     (iii) The measured  concentration of the test substance in each test
chamber just before the start of the  test and at all subsequent sampling

     (iv) In each test chamber at each observation period, the number of
dead and live test organisms, the percentage of organisms that died, and
the number of test organisms that  showed any abnormal  effects  in each
test chamber at each observation period.

     (v) The 48, 72 and 96-h LCSO's and their 95 percent confidence lim-
its. When sufficient data have been generated,  the 24-h LC50 value also.
These calculations should be made using the mean measured test substance

     (vi) The observed no-effect concentration (the highest concentration
tested at which there were no mortalities or abnormal behavioral or physio-
logical effects), if any.

     (vii) Methods and data for all chemical analyses of water quality and
test substance  concentrations,  including method validations and  reagent

     (9) A description of all circumstances that may have affected the qual-
ity or integrity of the data.

     (10) The names of the sponsor, study director, principal investigator,
names  of other scientists or professionals,  and the names of all supervisory
personnel involved in the study.

     (11) A description of the transformations, calculations, or operations
performed on the data, a summary and analysis  of the data,  and a statement
of the conclusions drawn from the analysis. Results of the analysis of data
should include the calculated LC50 value, 95 percent confidence limits,
slope of the transformed concentration-response line,  and the results  of
a goodness-of-fit test (e.g., X2 test).

     (12) The signed and dated reports prepared by any individual scientist
or other professional involved in the study, including each person who,
at the  request  or  direction of the  testing facility or  sponsor, conducted
an analysis  or  evaluation of  data or specimens from the study after data
generation was completed.

     (13) The locations where all specimens, raw data, and the final report
are stored.


(14) The statement prepared and signed by the quality assurance unit.