United States Prevention, Pesticides EPA712-C-96-038
Environmental Protection and Toxic Substances August 1996
Agency (7101)
&EPA Product Properties
Test Guidelines
OPPTS 830.7550
Partition Coefficient
(n-Octanol/Water),
Shake Flask Method
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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 pub-
lished 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. 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.).
Final Guideline Release: This document 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 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is available 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 830.7550 Partition coefficient (n-octanol/water), shake flask
method.
(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 materials used in developing this har-
monized OPPTS test guideline are the OPPT guideline under 40 CFR
796.1550 Partition Coefficient (n-Octanol/water), OPP guideline 63-11
Octanol/water partition coefficient (Pesticide Assessment Guidelines, Sub-
division D: Product Chemistry, EPA Report 540/9-82-018, October 1982),
and OECD guideline 107 Partition Coefficient (n-octanol/water).
(b) Introductory information—(1) Prerequisites. Suitable analytical
method; dissociation constant; water solubility; hydrolysis (preliminary
test).
(2) Coefficient of variation. The coefficient of variation on the mean
values reported by the participants of the 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 as a screening
test for bioaccumulation, it should be ascertained that the impurities in
the commercial product are of minor importance. Testing of partition coef-
ficient (w-octanol/water) cannot be used as a screening test in the case
of organometallic compounds.
(4) Recommendation. There is a high pressure liquid chroma-
tography (HPLC) method described in the references in paragraphs (f)(6),
(f)(7), and (f)(8) of this guideline which has not as yet been evaluated.
The convenience of this method recommends its investigation as an alter-
native method.
(5) Standard documents. The scientific literature in this field was
consulted extensively in developing this test guideline. It is based on the
references in paragraphs (f)(3) and (f)(9) of this guideline.
(c) Method—(1) Introduction, purpose, scope, relevance, applica-
tion, and limits of test. The partition coefficient of a substance between
water and a lipophilic solvent (w-octanol) is one model variable which
may be used to describe the transfer of a substance from the aquatic envi-
ronment into an organism and the potential bioaccumulation of the sub-
stance. Studies show a highly significant relationship between the partition
coefficient of different substances in the system water/w-octanol and their
bioaccumulation in fish described in the reference in paragraph (f)(l) of
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this guideline. It has also been shown to be a useful parameter in other
forms of biological activity (see the reference in paragraph (f)(2) of this
guideline).
(2) Definitions and units. The partition coefficient (P) is defined as
the ratio of the equilibrium concentrations (Q) of a dissolved substance
in a two-phase system consisting of two largely immiscible solvents. In
the case w-octanol and water
~~ L-n-
/L-
octanol-water
The partition coefficient (P) therefore is the quotient of two concentrations
and is usually given in the form of its logarithm to base ten (log P).
(3) Reference substances. The reference substances need not be em-
ployed 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 meth-
od is applied. The values presented below are not necessarily representa-
tive 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
Di(2-ethylhexyl)phthalate (OECD)
Hexachlorobenzene (OECD)
o-Dichlorobenzene (EEC)
Dibutyl phthalate (EEC)
Trichloroethylene (OECD)
Urea COECD1
1.3 x 105
3.6 x 105
5.1 x 103
1.3 x 104
2. Ox 103
6.2x10-2
P 2
r ow
(4.6 x 104-2
(1.1 x 105 -8
(1.5x 103 -2
(1.7x 103 -2
(5.2 x 102-3
C2.0x10-2-2
8x 105)
3x 105)
3 x 1 04)
8 x 1 04)
7 x 1 03)
.4x10-11
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 partition
coefficient, 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 preci-
sion of the partition coefficient, 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 partition coefficient expressed as their common
logarithms should fall within a range of ±0.3 log units.
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(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/L. The substance being
tested must not be water insoluble (mass concentration p > 10-6 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 partition coefficient becomes dependent upon the con-
centration of the solution. Because of the multiple equilibria involved, this
test guideline should not be applied to ionizable compounds without cor-
rections 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. Prelimi-
nary estimate of the partition coefficient. The size of the partition coeffi-
cient can be estimated either by means of a simple calculation (see para-
graph (f)(2) of this guideline) or by use of the solubilities of the test sub-
stance in the pure solvents (see references in paragraphs (f)(3) and (f)(5)
of this guideline). For this,
Pestimate = (saturation Cn-octanol)/(saturation Cwater)
Alternatively, it may be roughly determined by performing a simplified
preliminary test.
(2) Preparation of the solvents—(i) w-Octanol. The determination
of the partition coefficient should be carried out with analytical grade n-
octanol. Inorganic contaminants can be removed from commercial w-octa-
nol by washing with acid and base, drying, and distilling. More sophisti-
cated methods will be required to separate the w-octanol from organic con-
taminants with similar vapor pressure if they are present.
(ii) Water. Distilled water or water twice-distilled from glass or
quartz apparatus should be employed. (Note: Water taken directly from
an ion exchanger should not be used.)
(iii) Presaturation of the solvents. Before a partition coefficient 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 w-octanol or distilled water
each with a sufficient quantity of the other solvent for 24 hours on a me-
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chanical 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 partition coefficient (see para-
graph (d)(l)(i) of this guideline).
(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 w-octanol is added; and
in the third, half the volume of w-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
w-octanol with a mass concentration between 1 and 100 mg/mL. The actual
mass concentration of this stock solution should be precisely determined
before it is employed in the determination of the partition coefficient. This
solution should be stored under stable conditions.
(5) Test conditions. The test temperature should be kept constant
(+ 1 °C) and lie in the range of 20-25 °C.
(6) Performance of the test, (i) Establishment of the partition equi-
librium. 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 octa-
nol 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° 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.
(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-con-
trolled centrifuge is used, the centrifuge tubes should be reequilibrated at
the test temperature for at least 1 hour before analysis.
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(7) Analysis, (i) For the determination of the partition coefficient,
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 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 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) High pressure liquid chromatography.
(D) Back-extraction of the aqueous phase and subsequent gas chroma-
tography.
(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 partition coefficient and its man-
ner of determination.
(iv) Data on the analytical procedures used in determining concentra-
tions.
(v) The measured concentrations in both phases for each determina-
tion. (This means that a total of 12 concentrations will be reported).
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(vi) The weight of the test substance, the volume of each phase em-
ployed in each test vessel, and the total calculated amount of test substance
present in each phase after equilibration.
(vii) The calculated values of the partition coefficient (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 partition coefficient, 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. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Neely, W.B. et al. Partition Coefficients to Measure
Bioconcentration Potential of Organic Chemicals in Fish. Environmental
Science and Technology 8:1113 (1975).
(2) Leo, A. et al. Partition Coefficients and their Uses. Chemical Re-
views 71:525 (1971).
(3) Figge, K. et al. Die Bedeutung des Verteilungskoeffizienten fur
die Abschatzung der Bioakkumulation von Umweltchemikalien und
Methoden zu seiner Bestimmung. Report of the NATEC Gesellschaft fur
naturwissenschaftlich-technische Dienste mbH, Behringstr. 154, 2000
Hamburg 50.
(4) Hecker, E. Verteilungsverfahren im Laboratorium, Monographien
zur "Angewandten Chemie" und "Chemie-Ingenieur-Technik," No. 67,
Verlag Chemie, Weinheim 1955.
(5) Jubermann, O. Methoden der organischen Chemie 1/1:223 (1958).
(6) Miyake, K. and H. Terada, Direct measurements of partition coef-
ficients in an octanol-water system. Journal of Chromatography 157:386
(1978).
(7) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log
P for Organic Chemicals, EPA-600/3-78-049 (1978).
(8) Mirrless, M.S. et al., Direct measurement of octanol-water parti-
tion coefficient by high pressure liquid chromatography. Journal of Medic-
inal Chemistry 19:615 (1976).
(9) EPA Draft Guidance of September 8, 1978 (F-16).
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(10) Rekker, R.F. The Hydrophobic Fragmental Constant, Elsevier,
Amsterdam (1977).
(11) Konemann H. et al. Determination of log Poct values of
chlorosubstituted benzenes, toluenes, and anilines by high performance liq-
uid chromatography on ODS silica, Journal of Chromatography 178:559
(1979).
(12) Organization for Economic Cooperation and Development,
Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient
(n-octanol/water) (Flask-shaking Method), OECD, Paris, France.
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