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
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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 830.7370 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-
tion.
(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
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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
Ka=[H+][X-]/[HX]
or
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.
p-Nitrophenol
Benzole acid
D-Chloroaniline
7 15
4 19
4.15
pKa (1)
251
20
20
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)
20
20
20
(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)
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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.
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(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 =
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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).
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(2) Nelson, N.H. and Faust, S.D., "Acidic Dissociation Constants of
Selected Aquatic Herbicides," Environment Science Technology 3: II pp.
1186-1188(1969).
(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.
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