United States       Prevention, Pesticides     EPA712-C-96-036
          Environmental Protection    and Toxic Substances     August 1996
          Agency         (7101)
&EPA    Product Properties
          Test Guidelines
          OPPTS 830.7370
          Dissociation Constants
          in Water

     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 830.7370  Dissociation constants in water.
     (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
796.1370   Dissociation  constants  in  water,  OPP   guideline   63-10
Dissociation Constant (Pesticide Assessment  Guidelines, Subdivision D:
Product Chemistry, EPA Report 540/9-82-018, October 1982) and OECD
guideline 112 Dissociation Constants in Water.

     (b) Introductory information. (Titration method; spectrophotometric
method; conductometric method)

     (1) Prerequisites, (i) Suitable analytical method.

     (ii) Water solubility.

     (2) Guidance information, (i) Structural formula.

     (ii) Electrical conductivity for conductometric method.

     (3) Qualifying statements, (i)  All  test methods may be carried out
on pure or commercial grade substances. The possible effects of impurities
on results should be considered.

     (ii) The titration method  is not suitable for low solubility compounds
(see test solutions, under paragraph (c)(2)(ii)(A) of this guideline).

     (iii) The spectrophotometric method is only applicable to compounds
having appreciably different UV/VIS absorption spectra for the dissociated
and undissociated forms. This method may also be suitable for low solu-
bility compounds and for non-acid/base dissociations, e.g. complex forma-

     (iv) In cases where the Onsager  equation holds, the conductometric
method may be used, even at moderately low concentrations and even in
cases for non-acid/base  equilibria.

     (4) Standard documents. This test guideline is based on methods
given in the references listed in  paragraph (e) of this guideline and on
the Preliminary Draft Guidance for Premanufacture Notification EPA, Au-
gust 18, 1978.

     (c) Method—(1) Introduction, purpose,  scope, relevance, applica-
tion  and  limits of test—(i)  Dissociation of chemicals in water.  The
dissociation of a chemical in water is of importance in assessing its impact
upon the environment. It governs  the form of the  substance which in turn

      determines its behavior and transport.  It may affect the adsorption of the
      chemical on soils and sediments and absorption into biological cells.

           (ii) Definitions and units. Dissociation is the reversible splitting into
      two or more chemical species which may be ionic. The process is indicated
      generally by

                                 RX	> R+ + X-

      and the concentration equilibrium constant governing the reaction is

                                 K = [R+]  [X-]/[RX]

      For example,  in the particular case where R is hydrogen (the substance
      is an acid), the constant is



                                 pKa = pH - log [X ]/[HX]

           (iii) Reference substances.  (A) The following reference compounds
      need not be employed in all  cases when investigating a new substance.
      They are provided primarily so that calibration of the method may be per-
      formed from time to time and to offer the chance  to compare the results
      when another method is applied.

Benzole acid

7 15
4 19
pKa (1)
  1 No value is available for 20  °C, but it can be assumed that the variability of measurement results is
higher than the temperature dependence to be expected.

           (B) It would be useful to have a substance with several pK's as indi-
      cated in Principle of the Method, under paragraph (c)(l)(iii) of this guide-
      line. Such a compound could be:

Citric acid 	

(1) 3.14
(2) 4.77
(3) 6.39
pKa (8)
           (iv) Principle of the test method. The chemical process described
      is generally only  slightly temperature dependent in the  environmentally
      relevant temperature range. The determination of the dissociation constant
      requires  a  measure  of  the   concentrations  of  the  dissociated  and
      undissociated forms of the chemical substance.  From a knowledge of the
      stoichiometry of the dissociation reaction indicated in paragraph (c)(l)(ii)

of this guideline, the appropriate constant can be determined. In the par-
ticular case described in this guideline the substance is  behaving as an
acid or a base, and the determination is most conveniently done by deter-
mining the relative concentrations of ionized and un-ionized forms of the
substance and the pH of the solution. The relationship between these terms
is  given in the equation for pKa in paragraph (c)(l)(ii) of this guideline.
Some compounds  exhibit more than one dissociation constant and similar
equations can be  developed. Some of the methods described herein are
also suitable for non-acid/base dissociation.

     (v) Quality  criteria—(A)  Repeatability. The dissociation constant
should be replicated (a minimum of three determinations) to within ±0.1
log units.

     (B) [Reserved]

     (2)  Description of the test procedures—(i)  Determination  ap-
proaches. There  are two  basic  approaches  to the  determination of pKa.
One involves  titrating a known amount of substance with standard acid
or base,  as appropriate; the other involves  determining the  relative con-
centrations of the ionized and un-ionized forms and their pH dependence.

     (ii) Preparations. Methods based on those  principles may be classi-
fied as titration, spectrophotometric, and conductometric procedures.

     (A)   Test  solutions.   (7)   For  the  titration  method  and  the
conductometric method the chemical substance should be dissolved in dis-
tilled water. For  spectrophotometric  and other methods buffer solutions
are used. The  concentrations of the test substances  should not exceed the
lesser of 0.01  M or half the saturation concentration, and the purest avail-
able form of the substance should be employed in making up the solutions.
If the substance is only sparingly soluble, it may be dissolved in a small
amount  of a water-miscible solvent prior to adding to the concentrations
indicated above.

     (2) Solutions  should be  checked for the presence of emulsions using
a Tyndall beam, especially if a co-solvent has been used to enhance solu-
bility. Where  buffer  solutions are used, the buffer concentration should
not exceed 0.05 M.

     (B) [Reserved]

     (iii) Test conditions—(A) Temperature. (7) The temperature should
be controlled  to at least ±1  °C.  The determination should preferably be
carried out at 20 °C.

     (2) If a  significant temperature  dependence is suspected, the deter-
mination should be carried out at two or more different temperatures.  The
temperature intervals should be  10  °C in this case and the  temperature
control ±0.1 °C.

     (B)  Analyses. The method will be determined by the nature  of the
substance being tested. It must be sufficiently sensitive to allow determina-
tion of the different species at the test solution concentrations.

     (iv) Performance of the test—(A) Titration method. The test solu-
tion  is determined by titration with the standard base or acid solution as
appropriate, measuring the pH after each addition of titrant.  At least 10
incremental additions  should  be made before the equivalence point. If
equilibrium is reached sufficiently rapidly, a recording potentiometer may
be used.  For  this method both the total quantity of substance and its con-
centration need to be accurately known. Precautions must be taken to ex-
clude carbon dioxide.  Details of procedure, precautions, and calculation
are given in standard tests, e.g., references under paragraphs (e)(l) through
(e)(4) of this guideline.

     (B)  Spectrophotometric methods. A wavelength is found where the
ionized and un-ionized forms of the compound have appreciably different
extinction coefficients. The UV/VIS absorption spectrum is obtained from
solutions of constant concentration under a pH condition where the sub-
stance is essentially un-ionized and fully ionized and at  several intermedi-
ate pH's. This may be done  either by adding increments of concentrated
acid  (base) to a  relatively large  volume of a  solution  of the compound
in a  multicomponent buffer, initially at high (low) pH under paragraph
(e)(5) of this guideline, or by adding  equal volumes of a  stock solution
of the compound in, e.g., water, methanol, to constant volumes of various
buffer solutions  covering the  desired  pH range. From the pH  and
absorbance values at the chosen wavelength, a sufficient number of values
for the pKa are  calculated using data  from at least five pH's where the
compound is  at least 10 percent and less than 90 percent ionized. Further
experimental  details and method of calculation are given  in reference under
paragraph (e)(l)  of this guideline.

     (C)  Conductometric method. Using a  cell of small, known cell con-
stant,  the  conductivity  of an approximately  0.1 M  solution  of the
compound in conductivity water is measured. The conductivities of a num-
ber of accurately made dilutions  of this solution are also measured. (The
concentration is  halved each time, and the  series should cover at least an
order of  magnitude in concentration.) The limiting conductivity at infinite
dilution is  found by carrying out a  similar  experiment with the sodium
salt and  extrapolating. The degree of dissociation may then be calculated
from  the conductivity of each solution using the Onsager equation, and
hence  using the  Ostwald dilution law  the  dissociation  constant may be
calculated as

                           K = 
where C is the concentration in moles per liter and a is the fraction dis-
sociated. Precautions must be taken to exclude CC>2. Further experimental
details and method of calculation are given in standard texts and references
under paragraphs (e)(l), (e)(6), and (e)(7) of this guideline.

     (d) Data and  reporting—(1) Treatment of results—(i)  Titration
method. The pKa is calculated for 10 measured points on the titration
curve and the mean and  standard deviation of these pKa values are cal-
culated. A plot of pH versus volume of standard base or acid  should be
included along with a tabular presentation.

     (ii) Spectrophotometric  methods. The absorbance and pH are tab-
ulated from  each spectrum. At least five values for the pKa are  calculated
from the intermediate spectra data points, and the mean and standard devi-
ation of these results are also calculated.

     (iii) Conductometric method. The equivalent conductance, A, is cal-
culated for each acid concentration and for each concentration of a mixture
of one equivalent of acid, plus  0.98 equivalent of carbonate-free sodium
hydroxide. (The presence of excess  acid  is to prevent an excess of OH
due  to hydrolysis.) Values of I/A are plotted against Vc and A0  of the
salt  can be found by extrapolation to zero  concentration. AO of the acid
can  be calculated using  literature values for  H+  and Na+.  The pKa can
be calculated from a = Ai/Ao and Ka = oc2C/(l •  a) for each concentra-
tion. Better values for Ka can be obtained by making corrections for mobil-
ity  and activity.  The  mean and  standard deviations of the pKa  values
should be  calculated.

     (2) Test report, (i)  All raw data and  calculated pKa values should
be submitted together with method of calculation (preferably in a tabulated
format, such as suggested in paragraph (e)(l)  of this guideline) as should
the statistical parameters  described under paragraph (d)(l) of this  guide-
line. For titration methods, details of the standardization of titrants should
be given.

     (ii) For  spectrophotometric methods,  all spectra should be submitted.
For the conductometric method, details of the  cell constant determination
should be  reported. Information on technique used, analytical methods, and
the nature of any buffers used should be given.

     (iii) The test temperatures should be reported.

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

     (1) Albert, A. and Sergeant, E.P., lonization  Constants of Acids and
Bases. Wiley, Inc., New York (1962).

     (2) Nelson, N.H. and Faust, S.D., "Acidic Dissociation Constants of
Selected Aquatic Herbicides," Environment Science Technology 3: II pp.

     (3) American Society for Testing and Materials,  D-1293,  ASTM,
1916 Race St., Philadelphia, PA 19103 (latest annual index).

     (4) American Public  Health Association, Standard Methods for the
Examination of Water and Waste Water, Standard Method 242,  14th ed.,
American Public Health Association: Washington, DC (1976).

     (5) Clark, J. & Cunliffe, A.E., "Rapid Spectrophotometric Measure-
ment of lonisation Constants in Aqueous Solution," Chemical Index (Lon-
don  1973).

     (6) American Society for Testing and Materials,  D-1125,  ASTM,
1916 Race St. Philadelphia, PA 19103 (latest annual index).

     (7) American Public  Health Association, Standard Methods for the
Examination of Water and Waste Water. Standard Method 205,  14th ed.,
American Public Health Association: Washington, DC (1976).

     (8) Handbook of Chemistry and Physics, 60th ed.,  CRC-Press:  Boca
Raton, FL (1980).

     (9) Organization for Economic Cooperation and Development, Guide-
lines for The  Testing of Chemicals, OECD 112  Dissociation  Constants
in Water, OECD, Paris, France.