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
          Shake Flask Method

     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.).

     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,    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."

OPPTS 830.7550   Partition coefficient (n-octanol/water), shake flask
     (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

     (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

      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

           (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-

      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
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

8x 105)
3x 105)
3 x 1 04)
8 x 1 04)
7 x 1 03)
  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.

     (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-

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

     (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

     (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.

     (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

     (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-

     (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-

     (v) The measured concentrations in both phases for each determina-
tion. (This means that a total of 12 concentrations will be reported).

     (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

     (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).

    (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

    (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.