[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: 40CFR795.120]
[Page 59-63]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 795_PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart C_Provisional Environmental Effects Guidelines
Sec. 795.120 Gammarid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures subject to
environmental effects test regulations under the Toxic Substances
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003 (15 U.S.C. 2601 et
seq.)). This guideline describes a test to develop data on the acute
toxicity of chemicals to gammarids. The United States Environmental
Protection Agency (EPA) will use data from this test in assessing the
hazard of a chemical to aquatic organisms.
(b) Definitions. The definitions in section 3 of TSCA and in part
792 of this chapter, 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
prodding.
Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or
acclimation 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
organisms 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 concentration, 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) [Reserved]
(3) 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/1, etc.),
usually under static conditions.
(iii) A minimum of five gammarids should be exposed to each
concentration 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.
(4) Definitive test. (i) The purpose of the definitive test is to
determine 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
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and number of concentrations to which the organisms are exposed shall be
such that in 96 hours there is at least one concentration resulting in
mortality greater than 50 and less than 100 percent, and one
concentration causing greater than zero and less than 50 percent
mortality. An equal number of gammarids may be placed in two or more
replicate 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 determine 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
investigator should record the appearance of surface slicks,
precipitates, or material adhering to the sides of the test chambers.
(5) [Reserved]
(6) Analytical measurements--(i) Water quality analysis. The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and
particulate 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 chamber. Samples should not include any surface scum or material
dislodged from the bottom or sides. Samples shall be analyzed
immediately or handled and stored in a manner which minimizes loss of
test substance through microbial degradation, photogradation, chemical
reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the
concentration of dissolved test substance (that which passes through a
0.45 micron filter) shall be measured in each test chamber at least at
the beginning (zero-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 delivery system is observed.
(B) The analytical methods used to measure the amount of test
substance in a sample shall be validated before beginning the test. This
involves 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
concentration 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 test.
(C) An analytical method is not acceptable if likely degradation
products of the test substance give positive or negative interferences,
unless it is shown that such degradation products are not present in the
test chambers during the test.
(D) Among replicate test chambers, the measured concentrations shall
not
[[Page 61]]
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 LC50's and to plot all concentration-
response curves.
(d) Test conditions for definitive test--(1) Test species--(i)
Selection. (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 [deg]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
necessary it should be done as gently, carefully and quickly as
possible. During culturing and acclimation, gammarids shall be observed
carefully for signs of stress and mortality. Dead and abnormal
individuals shall be discarded.
(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:
(1) 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
temperature 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-hour light and 8-hour dark
photoperiod with a 15- to 30-minute transition period.
(B) Facilities should be well ventilated and free of fumes and
disturbances 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 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 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
[[Page 62]]
test substance delivery system shall be checked twice daily during a
test. The 24-hour 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 provide 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 acetone, 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 variation in hardness, acidity, alkalinity,
conductivity, TOC or COD, and particulate 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:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter......................... 20 mg/L
Total organic carbon (TOC) or.............. 2 mg/L
chemical oxygen demand (COD)............. 5 mg/L
Boron, fluoride............................ 100 ug/L
Un-ionized ammonia......................... 1 ug/L
Aluminum, arsenic, chromium, cobalt, 1 ug/L
copper, iron, lead, nickel, zinc.
Residual chlorine.......................... 3 ug/L
Cadmium, mercury, silver................... 100 ng/L
Total organophosphorus pesticides.......... 50 ng/L
Total organochlorine pesticides plus:
polychlorinated biphenyls (PCBs) or...... 50 ng/L
organic chlorine......................... 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
conductivity less than 1 micromho/cm is acceptable as the diluent for
making reconstituted water.
(C) The concentration of dissolved oxygen in the dilution water
shall be between 90 and 100 percent saturation. If necessary, the
dilution water can be aerated before the addition of the test substance.
All reconstituted water should be aerated before use.
(3) Test parameters. Environmental parameters during the test shall
be maintained as specified below:
(i) Water temperature of 18 1 [deg]C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation.
(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
requirements for the flow-through test will vary depending on the flow
rate of dilution water. The loading should not cause the dissolved
oxygen concentration 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
developed 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 information:
(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,
including 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
[[Page 63]]
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 rate.
(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
measurements.
(ii) If solvents are used, the name and source of the solvent, the
nominal 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
periods.
(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-hour LC50's and their 95 percent confidence
limits. When sufficient data have been generated, the 24-hour LC50 value
also. These calculations should be made using the mean measured test
substance concentrations.
(vi) The observed no-effect concentration (the highest concentration
tested at which there were no mortalities or abnormal behavioral or
physiological effects), if any.
(vii) Methods and data for all chemical analyses of water quality
and test substance concentrations, including method validations and
reagent blanks.
(9) A description of all circumstances that may have affected the
quality 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., chi-
square 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.
[52 FR 24462, July 1, 1987]