[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: 40CFR799.6755]
[Page 288-292]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart E_Product Properties Test Guidelines
Sec. 799.6755 TSCA partition coefficient (n-octanol/water), shake flask
method.
Source: 65 FR 78751, Dec. 15, 2000, unless otherwise noted.
(a) Scope--(1) Applicability. This section is intended to meet the
testing requirements of the Toxic Substances Control Act (TSCA) (15
U.S.C. 2601).
(2) Source. The source material used in developing this TSCA test
guideline is the Office of Prevention, Pesticides and Toxics (OPPTS)
harmonized test guideline 830.7550 (August 1996, final guideline). The
source is available at the address in paragraph (f) of this section.
(b) Introductory information--(1) Prerequisites. Suitable analytical
method, dissociation constant, water solubility, and hydrolysis
(preliminary test).
(2) Coefficient of variation. The coefficient of variation on the
mean values reported by the participants of the Organization for
Economic Coopertion and Development (OECD) Laboratory Intercomparison
Testing, Part I, 1979, appeared to be dependent on the chemicals tested;
it ranges from 0.17 to 1.03.
(3) Qualifying statements. This method applies only to pure, water
soluble substances which do not dissociate or associate, and which are
not surface active. In order to use the partition coefficient (P) as a
screening test for bioaccumulation, it should be ascertained that the
impurities in the commercial product are of minor importance. Testing of
P (n-octanol/water) cannot be used as a screening test in the case of
organometallic compounds.
(4) Alternative methods. High-pressure liquid chromatography (HPLC)
methods described in the references in paragraphs (f)(3), (f)(4), and
(f)(5) of this section may be considered as an alternative test method.
(c) Method--(1) Introduction, purpose, scope, relevance,
application, and limits of test. The P of a substance between water and
a lipophilic solvent (n-octanol) is one model variable which may be used
to describe the transfer of a
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substance from the aquatic environment into an organism and the
potential bioaccumulation of the substance. Studies show a highly
significant relationship between the P of different substances in the
system water/n-octanol and their bioaccumulation in fish described in
paragraph (f)(1) of this section.
(2) Definitions--Partition coefficient (P) is defined as the ratio
of the equilibrium concentrations (Ci) of a dissolved
substance in a two-phase system consisting of two largely immiscible
solvents. The P therefore is the quotient of two concentrations and is
usually given in the form of its logarithm to base 10 (log P). In this
case n-octanol and water:
Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.039
(3) Reference substances. The reference substances need not be
employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied. The values presented in table 1 of this
section are not necessarily representative of the results which can be
obtained with this test method as they have been derived from an earlier
version of the test guideline.
Table 1--Data for Reference Substances
------------------------------------------------------------------------
Tested substance \1\ Pow \2\
------------------------------------------------------------------------
Di(2-ethylhexyl)phthalate (OECD)....... 1.3 x 10\5\ (4.6 x 10\4\ - 2.8
x 10\5\)
Hexachlorobenzene (OECD)............... 3.6 x 10\5\ (1.1 x 10\5\ - 8.3
x 10\5\)
o-Dichlorobenzene European Economic 5.1 x 10\3\ (1.5 x 10\3\ - 2.3
Community (EEC). x 10\4\)
Dibutyl phthalate (EEC)................ 1.3 x 10\4\ (1.7 x 10\3\ - 2.8
x 10\4\)
Trichloroethylene (OECD)............... 2.0 x 10\3\ (5.2x10\2\-
3.7x10\3\)
Urea (OECD)............................ 6.2x10-2 (2.0x10-2--2.4x10-1)
------------------------------------------------------------------------
\1\ Substances not tested: Ethyl acetate, 4-methyl-2,4-pentanediol.
\2\ Total, mean, and range of mean values (in parentheses) submitted by
the participants of the OECD or EEC Laboratory Intercomparison
Testing.
(4) Principle of the test method. In order to determine a P,
equilibrium between all interacting components of the system must be
achieved, and the concentrations of the substances dissolved in the two
phases must be determined. A study of the literature on this subject
indicates that there are many different techniques which can be used to
solve this problem, i.e. the thorough mixing of the two phases followed
by their separation in order to determine the equilibrium concentration
for the substance being examined.
(5) Quality criteria--(i) Repeatability. In order to assure the
precision of the P, duplicate determinations are to be made under three
different test conditions, whereby the quantity of substance specified
as well as the ratio of the solvent volumes may be varied. The
determined values of the P expressed as their common logarithms should
fall within a range of 0.3 log units.
(ii) Sensitivity. The sensitivity of the method is determined by the
sensitivity of the analytical procedure. This should be sufficient to
permit the assessment of values of Pow up to 105 when the
concentration of the solute in either phase is not more than 0.01 mol/
Liter (L). The substance being tested must not be water insoluble (mass
concentration [rho] <= 10-6 gram (g)/L.
(iii) Specificity. The Nernst Partition Law applies only at constant
temperature, pressure, and pH for dilute solutions. It strictly applies
to a pure substance dispersed between two pure solvents. If several
different solutes occur in one or both phases at the same time, this may
affect the results. Dissociation or association of the dissolved
molecules result in deviations from the Nernst Partition Law. Such
deviations are indicated by the fact that the P becomes dependent upon
the concentration of the solution. Because of the multiple equilibria
involved, this test guideline should not be applied to
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ionizable compounds without corrections being made. The use of buffer
solutions in place of water should be considered for such compounds.
(iv) Possibility of standardization. This method can be
standardized.
(d) Description of the test procedure--(1) Preparations: Preliminary
estimate of the P. The size of the P can be estimated either by means of
calculation or by use of published solubilities of the test substance in
the pure solvents. Alternatively, it may be roughly determined by
performing a simplified preliminary test. For this:
Equation 2:
[GRAPHIC] [TIFF OMITTED] TR15DE00.040
(2) Preparation of the solvents--(i) n-Octanol. The determination of
the P should be carried out with analytical grade n-octanol. Inorganic
contaminants can be removed from commercial n-octanol by washing with
acid and base, drying, and distilling. More sophisticated methods will
be required to separate the n-octanol from organic contaminants with
similar vapor pressure if they are present.
(ii) Water. Distilled water or water twice-distilled from glass or
quartz apparatus should be employed. Water taken directly from an ion
exchanger should not be used.
(iii) Presaturation of the solvents. Before a P is determined, the
phases of the solvent system are mutually saturated by shaking at the
temperature of the experiment. For doing this, it is practical to shake
two large stock bottles of purified n-octanol or distilled water each
with a sufficient quantity of the other solvent for 24 hours on a
mechanical shaker, and then to let them stand long enough to allow the
phases to separate and to achieve a saturation state.
(3) Preparation for the test. The entire volume of the two-phase
system should nearly fill the test vessel. This will help prevent loss
of material due to volatilization. The volume ratio and quantities of
substance to be used are fixed by the following:
(i) The preliminary assessment of the P as discussed in paragraph
(d)(1) of this section).
(ii) The minimum quantity of test substance required for the
analytical procedure.
(iii) The limitation of a maximum concentration in either phase of
0.01 mol/L.
(iv) Three tests are carried out. In the first, the calculated
volume ratio is added; in the second, twice the volume of n-octanol is
added; and in the third, half the volume of n-octanol is added.
(4) Test substance. The test substance should be the purest
available. For a material balance during the test a stock solution is
prepared in n-octanol with a mass concentration between 1 and 100
milligram/milliliter (mg/mL). The actual mass concentration of this
stock solution should be precisely determined before it is employed in
the determination of the P. This solution should be stored under stable
conditions.
(5) Test conditions. The test temperature should be kept constant
( 1 [deg]C) and lie in the range of 20-25 [deg]C.
(6) Performance of the test--(i) Establishment of the partition
equilibrium. Duplicate test vessels containing the required, accurately
measured amounts of the two solvents together with the necessary
quantity of the stock solution should be prepared for each of the test
conditions. The n-octanol parts should be measured by volume. The test
vessels should either be placed in a suitable shaker or shaken by hand.
A recommended method is to rotate the centrifuge tube quickly through
180[deg] about its transverse axis so that any trapped air rises through
the two phases. Experience has shown that 50 such rotations are usually
sufficient for the establishment of the partition equilibrium. To be
certain, 100 rotations in 5 minutes are recommended.
[[Page 291]]
(ii) Phase separation. In order to separate the phases,
centrifugation of the mixture should be carried out. This should be done
in a laboratory centrifuge maintained at room temperature, or, if a non-
temperature-controlled centrifuge is used, the centrifuge tubes should
be reequilibrated at the test temperature for at least 1 hour before
analysis.
(7) Analysis. (i) For the determination of the P, it is necessary to
analyze the concentrations of the test substance in both phases. This
may be done by taking an aliquot of each of the two phases from each
tube for each test condition and analyzing them by the chosen procedure.
The total quantity of substances present in both phases should be
calculated and compared with the quantity of the substance originally
introduced.
(ii) The aqueous phase should be sampled by the following procedure
to minimize the risk of including traces of the n-octanol: A glass
syringe with a removable needle should be used to sample the water
phase. The syringe should initially be partially filled with air. Air
should be gently expelled while inserting the needle through the n-
octanol layer. An adequate volume of aqueous phase is withdrawn into the
syringe. The syringe is quickly removed from the solution and the needle
detached. The contents of the syringe may then be used as the aqueous
sample.
(iii) The concentration in the two-separated phases should
preferably be determined by a substance-specific method. Examples of
physical-chemical determinations which may be appropriate are:
(A) Photometric methods.
(B) Gas chromatography.
(C) HPLC.
(D) Back-extraction of the aqueous phase and subsequent gas
chromatography.
(e) Data and reporting--(1) Treatment of results. The reliability of
the determined values of P can be tested by comparison of the means of
the duplicate determinations with the overall mean.
(2) Test report. The following should be included in the report:
(i) Name of the substance, including its purity.
(ii) Temperature of the determination.
(iii) The preliminary estimate of the P and its manner of
determination.
(iv) Data on the analytical procedures used in determining
concentrations.
(v) The measured concentrations in both phases for each
determination. This means that a total of 12 concentrations must be
reported.
(vi) The weight of the test substance, the volume of each phase
employed in each test vessel, and the total calculated amount of test
substance present in each phase after equilibration.
(vii) The calculated values of the P and the mean should be reported
for each set of test conditions as should the mean for all
determinations. If there is a suggestion of concentration dependency of
the P, this should be noted in the report.
(viii) The standard deviation of individual P values about their
mean should be reported.
(ix) The mean P from all determinations should also be expressed as
its logarithm (base 10).
(f) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available from the TSCA Nonconfidential Information
Center, Rm. NE-B607, Environmental Protection Agency, 401 M St., SW.,
Washington, DC, 12 noon to 4 p.m., Monday through Friday, excluding
legal holidays.
(1) Neely, W.B. et al. Partition Coefficients to Measure
Bioconcentration Potential of Organic Chemicals in Fish. Environmental
Science and Technology 8:1113 (1974).
(2) Leo, A. et al. Partition Coefficients and Their Uses. Chemical
Reviews 71:525 (1971).
(3) Miyake, K. and H. Terada, Direct measurements of partition
coefficients in an octanol-water system. Journal of Chromatography
157:386 (1978).
(4) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log P
for Organic Chemicals, EPA-600/3-78-049 (1978).
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(5) Mirrless, M.S. et al., Direct measurement of octanol-water
partition coefficient by high pressure liquid chromatography. Journal of
Medicinal Chemistry 19:615 (1976).
(6) EPA Draft Guidance of September 8, 1978 (F-16).
(7) Konemann H. et al. Determination of log Poct values
of chlorosubstituted benzenes, toluenes, and anilines by high
performance liquid chromatography on ODS silica, Journal of
Chromatography 178:559 (1979).
(8) Organization for Economic Cooperation and Development,
Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient
(n-octanol/water) (Shake Flask Method, Adopted 27 July 1995), OECD,
Paris, France.