United States      Prevention, Pesticides     EPA712-C-96-040
         Environmental Protection    and Toxic Substances     August 1996
         Agency        (7101)
&EPA    Product Properties
          Test Guidelines
          OPPTS 830.7570
          Partition Coefficient
          Estimation By Liquid

     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 830.7570  Partition coefficient (n-octanol/water), estimation
by liquid chromatography.
     (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
796.1570  Partition  Coefficient (n-Octanol/water)—Estimation by liquid
chromatography, OPP guideline 63-11 OctanoI/water partition coefficient
(Pesticide Assessment Guidelines, Subdivision D: Product Chemistry, EPA
Report 540/9-82-018, October 1982), and OECD guideline  117  Partition
Coefficient (n-octanol/water), High Performance Liquid Chromatography
(HPLC) Method.

     (b) Introduction. (1)  The partition  coefficient (P) is defined as the
ratio of the equilibrium  concentrations of a dissolved substance in a two-
phase system consisting of two largely immiscible solvents. The partition
coefficient being the quotient of two concentrations, is  dimensionless and
is usually given in  the form  of its logarithm to base ten. In the case  of
w-octanol and water,

                           "ow ~~ L-n-octanol/L-water

     (2) Pow is a key parameter  in studies of the  environmental fate  of
chemical substances. A highly-significant relationship between the Pow  of
substances and their bioaccumulation  in fish has been  shown. It has also
been shown that Pow is a useful parameter in the prediction of adsorption
on soil  and sediments and for establishing quantitative structure-activity
relationships for a wide range  of biological effects.

     (3) POW values  in the range log Pow between -2 and 4 can be experi-
mentally determined by the shake-flask method (OPPTS 830.7550). Pow
values in the range  log Pow between 0 and 6 can be estimated using high
performance  liquid chromatography  (HPLC)  under  paragraphs (f)(l)
through (f)(4) of this guideline. The HPLC method requires a preliminary
estimation of Pow, generally done through calculation. Calculation methods
are briefly discussed under paragraph (d)(l)(i) of this guideline.

     (c) Principle of the method. (l)(i) HPLC is performed on analytical
columns packed with a commercially available solid phase containing long
hydrocarbon chains  (e.g. Cg, Cig) chemically bound onto silica. Chemicals
injected onto such  a column move along it by  partitioning between the
mobile solvent phase and the hydrocarbon stationary phase. The chemicals
are retained in proportion to their hydrocarbon-water partition coefficient,
with water-soluble  chemicals eluted first and oil-soluble chemicals last.
This enables the relationship between the retention time  on a reverse-phase
column  and the w-octanol/water partition coefficient to be established. The

partition coefficient is deduced from the  capacity factor k, given by the

                           k = (tR - t0)/to

where  tR  is the retention time of the test substance, and t0 is the dead-
time, i.e. the average time a solvent molecule needs to pass the column.
Quantitative analytical methods are not required and only the determina-
tion of retention times is necessary.  If standard reference compounds are
available and standard experimental conditions are used, the HLPC method
can be performed faster than shake-flask method under OPPTS 830.7550.

    (ii) The HPLC method enables  partition coefficients to be estimated
in the  log  Pow range between 0  and 6. The method is not applicable to
strong  acids and bases, metal complexes,  substances which react with the
eluent,  or  surface-active  agents. Measurements  should   be  made  on
ionizable substances  in their nonionized form (free acid or free base) only
by using an appropriate  buffer with a pH below the pK for a free acid
or above  the pK for a free base (e.g.  phosphoric acid  for pH  =  2 and
0.01-0.02 M phosphate buffer for pH = 7.5).

    (iii) The HPLC  method is less sensitive to the presence of impurities
in the  test substance  than the shake-flask method. Nevertheless,  in some
cases  impurities can  make the interpretation  of the  results difficult due
to uncertainty in peak assignments. For mixtures which result in  an unre-
solved band, upper and lower limits of log P should be  stated (see para-
graph (f)(3) of this guideline).

    (2) Information on the test substance.  The structural formula and
the dissociation constant  should be known before using the method. Infor-
mation on solubility and hydrolysis characteristics is useful.

    (3) Repeatability and accuracy. In order to increase the confidence
in the  measurement,  duplicate determinations must be made. The values
of log  POW derived from the different measurements should fall  within a
range  of ±0.1  log units. An  interlaboratory comparison test has shown
that with the HPLC method log Pow values can be obtained to within ±0.5
units of the shake-flask values (under paragraph (f)(5) of this guideline).
Other  comparisons can be found under paragraphs  (f)(3),  (f)(4),  (f)(6),
(f)(7),  and (f)(8) of this guideline. Correlation graphs based on structurally
related reference compounds give the most accurate results (under para-
graph (f)(9) of this guideline)

    (4) Reference compounds, (i) In order to correlate the measured ca-
pacity  factor k of a  compound with its Pow, a calibration graph using at
least 6 points has to be established. It is up to the user to select the appro-
priate  reference compounds.  It is preferable that these  should  be struc-
turally related to the  test substance.  Whenever possible, at  least one ref-
erence compound should have a Pow above that of the test substance, and

another a Pow below that of the test substance.  For log Pow values below
4, the calibration can be based on data obtained by the shake flask method.
For log  POW values above  4, the calibration can  be based on literature
values if they correspond to  calculated values.

    (ii)  Extensive  lists of log Pow values for many groups of chemicals
are  available (under paragraphs (f)(10)  and (f)(ll) of this guideline).  If
data on the partition coefficients of structurally related compounds are not
available, a more general calibration, established with other reference com-
pounds, may be used.  Recommended  reference  compounds and their Pow
values are listed in the  following table  1.  For ionizable  substances the
values given apply to  the nonionized  form.  The values were checked for
plausibility and quality during an interlaboratory comparison test.

              Table 1.—Recommended  Reference Compounds
Reference substance
Benzyl alcohol
Phenoxyacetic acid
2 4-Dinitrophenol
4-Methylbenzyl alcohol 	
3-Nitrobenzoic acid
Cinnamic alcohol
Benzole acid 	
c/s-Cinnamic acid 	
frans-Cinnamic acid
Methyl benzoate
3-Methylbenzoic acid
Ethyl benzoate 	
r ow
1 0
1 1
1 4
1 5
1 6
1 7
1 8
1 9
1 9
2 1
2 1

3 12


10 17


Reference substance
3-Chlorobenzoic acid 	
2 3-Dichloroaniline
Allyl phenyl ether 	
4-Phenyl phenol 	
1 ,4-Dichlorobenzene 	
Phenyl benzoate
Benzyl benzoate 	
2 4-Dinitro-6-sec-butyl phenol
1 ,2,4-Trichlorobenzene 	
Dodecanoic acid
Diphenyl ether 	
2 6-Diphenylpyridine

r ow
4 1






     (d) Description of the method—(1)  Preliminary  estimate of the
partition coefficient. The partition coefficient of the test substance is esti-
mated preferably by using a calculation method or, where appropriate, by
using the ratio of the solubilities of the test substance in the pure solvents
(under paragraph (f)(12) of this guideline).

     (i) POW calculation methods. This discussion provides a short intro-
duction to  the  calculation  of Pow.  For further information the reader is
referred to  paragraphs (f)(15) and (f)(16) of this guideline. Calculated val-
ues of POW  are used for:

     (A) Deciding which experimental method to use: Shake flask method
for log POW between -2 and 4 and HPLC method for log Pow between
0 and 6.

     (B) Selecting conditions to be used in HPLC (reference compounds,
methanol/water ratio).

     (C) Checking the plausibility of values obtained through experimental

     (D) Providing an estimate when experimental methods cannot be ap-

     (ii) Principle of calculation methods. Calculation methods are based
on the theoretical fragmentation of the molecule into suitable substructures
for which reliable log Pow increments are known.  The log Pow is obtained
by   summing  the fragment  values   and  the   correction  terms  for
intramolecular  interactions. Lists  of fragment constants and correction
terms are available under paragraphs (f)(15) through  (f)(20) of this guide-
line; some  are  regularly updated (under paragraph (f)(17) of this guide-

     (iii) Reliability of calculated values. In general, the reliability of cal-
culation methods  decreases  as the complexity of the compound under study
increases. In the  case of simple molecules of low molecular weight and
with one or two functional  groups, a deviation of 0.1 to 0.3  log Pow units
between the results of the different fragmentation methods and the meas-
ured value  can  be expected. The margin of error will depend on the reli-
ability  of   the  fragment   constants  used,   the  ability  to recognize
intramolecular  interactions  (e.g.  hydrogen bonds)  and the correct use of
correction terms.  In the case of ionizing compounds the charge and degree
of ionization must be  taken into  consideration (under paragraph (f)(24)
of this guideline).

     (iv) Fujita-Hansch 7i-method. The hydrophobic substituent constant,
71, originally introduced under paragraph (f)(21) of this  guideline, is de-
fined as:

                       TCX = log Pow(PhX) - log Pow(PhH),

where  PhX is an aromatic  derivative and PhH the parent compound.  For

        = log P0w(C6H5Cl) - log P0w(C6H6) = 2.84 - 2.13 = 0.71

The 7i-method is primarily of interest for aromatic compounds. Values of
7i for a large number of substituents are available (see paragraphs (f)(18)
and (f)(19) of this guideline).

     (v) Rekker  method. Using  the  Rekker  method  (under paragraph
(f)(22) of this guideline) the log Pow value is calculated as:

               log Pow =2_,aifi + /1(interactionterms}
where ai is the number of a  given fragment present in the molecule and
fi is the log POW increment  of the  fragment.  The interaction terms can
be expressed as an  integral multiple of one single constant Cm (so-called
"magic constant").  The fragment constants fi and Cm have  been deter-
mined from  a list of 1,054 experimental  Pow  values of 825  compounds
using multiple  regression analysis  (under  paragraphs (f)(20)  and (f)(22)
of this  guideline). The determination  of the interaction terms is carried
out according to  set rules (under  paragraphs (f)(20), (f)(22),  and (f)(23)
of this guideline).

     (vi) Hansch-Leo method. Using the Hansch and Leo method (under
paragraph (f)(18) of this  guideline), the log Pow value  is calculated as:
where  fi is a fragment constant, Fj  a correction term (factor),  ai and bj
the corresponding frequency of occurence. Lists of atomic and group frag-
mental values and of correction terms Fj  were derived by trial  and error
from experimental Pow values.  The correction terms  have been divided
into several different classes  (under paragraphs (f)(15) and (f)(18) of this
guideline). Software packages have been  developed to take  into account
all the rules and correction terms  (under paragraph (f)(17) of this guide-

    (vii) Combined method. The calculation of log Pow of complex mol-
ecules  can be considerably improved, if the molecule is dissected into larg-
er substructures for which reliable log Pow  values are available, either from
tables  under paragraphs (f)(3) and (f)(4) of this guideline or by existing
measurements.   Such   fragments   (e.g.   heterocycles,   anthraquinone,
azobenzene)  can  then be  combined with the Hansch-7i-values or with
Rekker or Leo fragment constants.

    (viii) Remarks. (A) The calculation  methods are only applicable to
partly  or fully ionized compounds when the necessary correction factors
are taken into account.

     (B)  If the existence  of intramolecular hydrogen  bonds can be  as-
sumed, the corresponding correction terms  (approximately +0.6 to +1.0
log POW units) must be added (under paragraph (f)(15) of this guideline).
Indications on the presence of such bonds  can be obtained  from stereo
models or spectroscopic data.

     (C)  If several tautomeric forms are possible,  the most  likely form
should be used as the basis of the calculation.

     (D)  The revisions of lists of fragment constants should  be followed

     (2) Apparatus. A liquid-phase chromatograph, fitted with a pulse-
free pump and a suitable detection device is required. The use of an injec-
tion valve with  injection loops is  recommended. The presence of polar
groups in the stationary phase may seriously impair the performance  of
the HPLC column.  Therefore, stationary phases  should have a minimal
percentage of polar groups (see paragraph (f)(13) of this guideline). Com-
mercial microparticulate reverse-phase packings or ready-packed columns
can be used. A  guard column may be positioned  between the injection
system and the analytical column.

     (3) Mobile  phase, (i) HPLC-grade methanol and  distilled water  are
used to prepare the eluting solvent, which is degassed before use. Isocratic
elution should be employed. Methanol/water ratios with a minimum water
content of 25 percent should be used. Typically a 3:1 (v/v)  methanol/water
mixture is satisfactory for  eluting  compounds with a  log P  of 6 within
an hour,  at a flow rate of 1 mL/min. For compounds with a  log P above
6 it may be necessary to  shorten the elution time (and those of the ref-
erence compounds) by decreasing the polarity of the mobile phase or  the
column length.

     (ii) The test substance and the reference compounds should be soluble
in the mobile phase in sufficient concentration to  allow  their  detection.
Additives may be used with the methanol/water mixture in exceptional
cases only, since they will change the  properties  of the column. In these
cases a  separate column of the same type should be used. If methanol/
water is not appropriate, other organic solvent/water mixtures can be used,
e.g. ethanol/water and acetonitrile/water.

     (iii) The pH of the  eluent is critical  for ionizable  compounds. It
should be within the operating pH range of the column, usually between
2 and 8.  Buffering is recommended. Care must be taken to avoid salt pre-
cipitation and column deterioration which occur with some organic phase/
buffer mixtures.  HPLC measurements with silica-based stationary phases
above pH 8 are  not  advisable since the use of an alkaline mobile phase
may cause rapid deterioration in the performance of the column.

     (4) Solutes. The test and reference compounds should be the purest
available.  Compounds to be used for test or calibration purposes are dis-
solved in the mobile phase if possible.

     (5) Test conditions. The temperature during the measurements should
not vary by more than + 2 K.

     (6) Determination of dead time to. The dead time to can be meas-
ured by using unretained organic compounds (e.g. thiourea  or formamide).
It can  also be derived from the retention times measured for a set of ap-
proximately seven  members of a  homologous series (e.g.  w-alkylmethyl
ketones) (under paragraph (f)(14)  of this guideline). The retention times
tR(nc + i) are plotted against tR(nc), where nc is the number of carbon atoms.
A straight line,

                tR(nc + 1) = AtR(nc) + (1 ~ A)to

is  obtained, where  A, representing k(nc + i)/k(nc),  is constant. The dead
time to is obtained from the intercept (1 - A)to and the slope A.

     (7) Calibration  graph. The next step is to plot a correlation graph
of log k versus log  P for appropriate reference compounds  with log P
values near the value expected for the  test substance. In practice, from
5 to 10 reference compounds are injected  simultaneously. The retention
times are  determined, preferably on a  recording integrator linked  to the
detection  system. The corresponding logarithms of the  capacity factors,
log k,  are calculated  and plotted as a function of  log P. The calibration
is performed at regular intervals, at least once daily, so  that account can
be taken of possible changes in column performance.

     (8) Determination of the Pow of the  test substance. The test sub-
stance  is injected in the  smallest quantity possible.  The  retention time is
determined in duplicate.  The partition coefficient of the  test substance is
obtained by interpolation of the calculated capacity factor  on the calibra-
tion graph. Extrapolation is necessary for very low and very high partition
coefficients. In these cases, special attention must be given to the con-
fidence limits of the regression line.

     (e) Test report. The following should be included in the report:

     (1) Test and reference substances, and their purity.

     (2) Description of equipment and operating conditions: Analytical col-
umn, guard column.

     (3) Mobile phase, means of detection, temperature range, pH.

     (4) Elution profiles.

     (5) Dead time and how it was measured.

     (6) Quantities of test and references substances introduced in the col-

     (7) Retention data and literature log P values for reference compounds
used in calibration.

     (8) Details on fitted regression line (log k versus log P).

     (9) Preliminary estimate of the  partition coefficient  and the method
used, and, if a calculation method was used, its full description including
identification of the data base and detailed information on the choice of

     (10) Average retention data and interpolated log P value for the test

     (f) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1) Eadsforth, C.V. and P. Moser. Chemosphere 12:1459 (1983).

     (2) Eadsforth, C.V. Pesticide Science 17:311 (1986).

     (3) Ellgehausen, H. et al. Pesticide Science 12:219 (1981).

     (4) McDuffie, B. Chemosphere 10:73 (1981).

     (5) Klein W. et al. Chemosphere 17:361 (1988).

     (6) Renberg, L.O.  et al. Chemosphere 9:683 (1980).

     (7) Hammers, W.E. et al. Journal of Chromatography 247:1 (1982).

     (8) Haky, J.E. and A.M. Young. Journal of Liquid Chromatography
7:675 (1984).

     (9) Fujisawa, S. and E.  Masuhara. Journal of Biomedical Materials
Research 15:787(1981).

     (10) Hansch, C. and A.J. Leo. Substituent Constants for Correlation
Analysis in Chemistry and Biology, Wiley, New York (1979).

     (11) Pomona College Medical Chemistry Project,  C.  Hansch,  Chair-
man; A.J. Leo, Director. Log P and Parameter Database: A tool for the
quantitative prediction of bioactivity.  Available  from Pomona College,
Claremont, CA 91711 (1982).

     (12)  Jubermann,  O. Verteilen  und Extrahieren,  in Methoden der
Organischen  Chemie  (Houben   Weyl),   Band   I/I,   Allgemeine
laboratoriumpraxis  (edited by E. Miiller),  pp.  223-239,  Georg Thieme
Verlag, Stuttgart (1958).


     (13) Rekker, R.F. and H.M. de Kort, European Journal of Medicinal
Chemistry 14:479 (1979).

     (14) Berendsen, G.E. et al. Journal of Liquid Chromatography 3:1669

     (15) Lyman, W.J. W.F. Reehl, and D.H. Rosenblatt (Eds.), Handbook
of  Chemical Property Estimation  Methods, McGraw-Hill,  New  York

     (16) Dunn, W.J., J.H. Block and R.S. Pearlman (Ed.), Partition Coef-
ficient, Determination and Estimation, Pergamon, Elmsford (New York)
and Oxford (1986).

     (17) Pomona College, Medicinal Chemistry Project,  Claremont, CA
91711, USA, Log P Database and Medical  Chemistry Software (Program

     (18) Hansch, C. and A.J. Leo,  Substituent Constants for Correlation
Analysis in Chemistry and Biology Wiley, New York (1979).

     (19) Leo, A. et al. Chemical Reviews 71:525 (1971).

     (20) Rekker, R.F. and H.M. de Kort. European Journal of Medicinal
Chemistry 14:479 (1979).

     (21)  Fujita,  T.  et al.  Journal  of the  American Chemical Society

     (22)   Rekker,   R.F.  The  Hydrophobic  Fragmental   Constant,
Pharmacochemistry Library, vol. 1, Elsevier, New York (1977).

     (23) Eadsforth, C.V. and P. Moser. Chemosphere 12:1459 (1983).

     (24) Scherrer, R.A. American  Chemical Society Symposium Series
no. 255, p. 225, American Chemical  Society, Washington, DC (1984).

     (25)  Organization for  Economic  Cooperation and Development,
Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient
(n-octanol/water), High Performance  Liquid Chromatography (HPLC)
Method, OECD, Paris, France.