[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.6756]
[Page 292-303]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart E_Product Properties Test Guidelines
Sec. 799.6756 TSCA partition coefficient (n-octanol/water), generator
column method.
(a) Scope--(1) Applicability. This section is intended to meet the
testing requirements of the Toxic Substances Control Act (TSCA) (15
U.S.C. 2601).
(2) Source. The source material used in developing this TSCA test
guideline is the Office of Pollution Prevention, Pesticides and Toxic
Substances (OPPTS) harmonized test guideline 830.7560 (August 1996,
final guideline). This source is available at the address in paragraph
(e) of this section.
(b)(1) Purpose. (i) The measurement and estimation of the n-octanol/
water partition coefficient (Kow), has become the cornerstone
of a myriad of structure-activity relationships (SAR) property. The
coefficient has been used extensively for correlating structural changes
in drugs with changes observed in biological, biochemical, or toxic
effects. These correlations are then used to predict the effect of a new
drug for which a Kow could be measured.
(ii) In the study of the environmental fate of organic chemicals,
the Kow has become a key parameter. Kow is
correlated to water solubility, soil/sediment sorption coefficient, and
bioconcentration and is important to SAR.
(iii) Of the three properties that can be estimated from
Kow, water solubility is the most important because it
affects both the fate and transport of chemicals. For example, highly
soluble chemicals become quickly distributed by the hydrologic cycle,
have low-sorption coefficients for soils and sediments, and tend to be
more easily degraded by microorganisms. In addition, chemical
transformation processes such as hydrolysis, direct photolysis, and
indirect photolysis (oxidation) tend to occur more readily if a compound
is soluble.
(iv) Direct correlations between Kow and both the soil/
sediment sorption coefficient and the bioconcentration factor are to be
expected. In these cases, compounds that are more soluble in n-octanol
(more hydrophobic and lipophilic) would be expected to partition out of
the water and into the organic portion of soils/sediments and into
lipophilic tissue. The relationship between Kow and the
bioconcentration factor, are the principal means of estimating
bioconcentration factors. This relationship is discussed in the
reference listed in paragraph (e)(14) of this section. These factors are
then used to predict the potential for a chemical to accumulate in
living tissue.
(v) This section describes a method for determining the
Kow based on the dynamic coupled column liquid
chromatographic (DCCLC) technique, a technique commonly referred to as
the generator column method. The method described herein can be used in
place of the standard shake-flask method specified in Sec. 799.6755 for
compounds with a log10Kow greater than 1.0.
(2) Definitions. The following definitions apply to this section.
Extractor column is used to extract the solute from the aqueous
solution produced by the generator column. After extraction onto a
bonded chromatographic support, the solute is eluted with a solvent/
water mixture and subsequently analyzed by high-performance liquid
chromatography (HPLC), gas chromatography (GC), or any other analytical
procedure. A detailed description of the preparation of the extractor
column is given in paragraph (c)(1)(i) of this section.
Generator column is used to partition the test substance between the
n-octanol and water phases. The column in figure 1 in paragraph
(c)(1)(i)(A)(2) of
[[Page 293]]
this section is packed with a solid support and is coated with the test
substance at a fixed concentration in n-octanol. The test substance is
eluted from the column with water and the aqueous solution leaving the
column represents the equilibrium concentration of the test substance
that has partitioned from the n-octanol phase into the water phase.
Preparation of the generator column is described in paragraph (c)(1)(i)
of this section.
n-Octanol/water partition coefficient (Kow) is defined as
the ratio of the molar concentrations of a chemical in n-octanol and
water, in dilute solution. The coefficient Kow is a constant
for a given chemical at a given temperature. Since Kow is the
ratio of two molar concentrations, it is a dimensionless quantity.
Sometimes Kow is reported as the decadic logarithm
(log10Kow). In this equation, Coctanol
and Cwater are the molar concentration of the solute in n-
octanol and water, respectively, at a given temperature. This test
procedure determines Kow at 25 0.05
[deg]C. The mathematical statement of Kow is:
Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.041
Response factor (RF) is the solute concentration required to give a
one unit area chromatographic peak or one unit output from the HPLC
recording integrator at a particular recorder and detector attenuation.
The factor is required to convert from units of area to units of
concentration. The determination of the RF is given in paragraph
(c)(3)(iii)(C)(2) of this section.
Sample loop is a \1/16\ inch (in) outside diameter (O.D.) (1.6
millimeter (mm)) stainless steel tube with an internal volume between 20
and 50 [micro]L. The loop is attached to the sample injection valve of
the HPLC and is used to inject standard solutions into the mobile phase
of the HPLC when determining the RF for the recording integrator. The
exact volume of the loop must be determined as described in paragraph
(c)(3)(iii)(C)(1) of this section when the HPLC method is used.
(3) Principle of the test method. (i) This test method is based on
the DCCLC technique for determining the aqueous solubility of organic
compounds. The development of this test method is described in the
references listed in paragraphs (e)(6), (e)(12), and (e)(19) of this
section. The DCCLC technique utilizes a generator column, extractor
column, and HPLC coupled or interconnected to provide a continuous
closed-flow system. Aqueous solutions of the test compound are produced
by pumping water through the generator column that is packed with a
solid support coated with an approximately 1.0% weight/weight (w/w)
solution of the compound in n-octanol. The aqueous solution leaving the
column represents the equilibrium concentration of the test chemical
which has partitioned from the n-octanol phase into the water phase. The
compound is extracted from the aqueous solution onto an extractor
column, then eluted from the extractor column with a solvent/water
mixture and subsequently analyzed by HPLC using a variable wavelength
ultraviolet (UV) absorption detector operating at a suitable wavelength.
Chromatogram peaks are recorded and integrated using a recording
integrator. The concentration of the compound in the effluent from the
generator column is determined from the mass of the compound (solute)
extracted from a measured volume of water (solvent). The Kow
is calculated from the ratio of the molar concentration of the solute in
the 1.0% (w/w) n-octanol and molar concentration of the solute in water
as determined using the generator column technique.
(ii) Since the HPLC method is only applicable to compounds that
absorb in the UV, an alternate GC method, or any other reliable
quantitative procedure must be used for those compounds that do not
absorb in the UV. In the GC method the saturated solutions produced in
the generator column are extracted using an appropriate organic solvent
that is subsequently injected into the GC, or any other suitable
analytical device, for analysis of the test compound.
(4) Reference chemicals. (i) Columns 2, 3, 4, and 5 of table 1 in
paragraph (b)(4)(ii) of this section list the experimental values of the
decadic logarithm of the n-octanol/water partition coefficient
(log10Kow) at 25 [deg]C for a number of organic
chemicals as obtained from the
[[Page 294]]
scientific literature. These values were obtained by any one of the
following experimental methods: Shake-flask; generator column; reverse-
phase HPLC; or reverse-phase thin-layer chromatography, as indicated in
the footnotes following each literature citation. The estimation method
of Hawker and Connell as described in paragraph (e)(8) of this section,
correlates log10Kow with the total surface area of
the molecule and was used to estimate log10Kow for
biphenyl and the chlorinated biphenyls. These estimated values are
listed in column 7 of table 1 in paragraph (b)(4)(ii) of this section.
Recommended values of log10Kow were obtained by
critically analyzing the available experimental and estimated values and
averaging the best data. These recommended values are listed in column 8
of table 1 in paragraph (b)(4)(ii) of this section.
(ii) The recommended values listed in table 1 of this section have
been provided primarily so that the generator column method can be
calibrated and to allow the chemical laboratory the opportunity to
compare its results with these values. The testing laboratory has the
option of choosing its reference chemicals, but references must be given
to establish the validity of the measured values of
log10Kow.
Table 1--n-Octanol/Water Partition Coefficient at 25 [deg]C for Some Reference Compounds
--------------------------------------------------------------------------------------------------------------------------------------------------------
Experimental log10Kow Estimated log10Kow
-----------------------------------------------------------------------
Chemical Generator Hansch Recommended
Hansch and Column Banerjee\2\ Other and Hawker and log10Kow
Leo\1\ Method values Leo\3\ Connell\4\
--------------------------------------------------------------------------------------------------------------------------------------------------------
Ethyl acetate................................................. 0.73, 0.66 \5\0.68 -- -- 0.671 -- \17\0.685
1-Butanol..................................................... 0.88, 0.89, \5\0.785 -- -- 0.823 -- \23\0.852
0.32, 0.88
1-Pentanol.................................................... 1.28, 1.40 \5\1,53 -- -- 1.35 -- \17\1.39
Nitrobenzene.................................................. 1.85, 1.88, \5\1.85 1.83 \6\1.82 1.89 -- \17\1.84
1.79
Benzene....................................................... 2.15, 2.13 -- 2.12 -- 2.14 -- \17\2.14
Trichloroethylene............................................. 2.29 \5\2.53 2.42 -- 2.27 -- \17\2.38
Chlorobenzene................................................. 2.84, 2.46 \7\2.98 -- \8\2.84 2.86 -- \18\2.80
o-Dichlorobenzene............................................. 3.38 \7\3.38 3.40 \8\3.38 3.57 -- \17\3.42
n-Propylbenzene............................................... 3.66, 3.66, \5\3.69 -- -- 3.85 -- \17\3.69
3.68, 3.57
Biphenyl...................................................... 3.95, 4.17, \7\3.67, 4.04 \6\3.75 4.03 4.09 \17\3.96
4.09, 4.04 \9\3.89,
\10\3.79
2-Chlorobiphenyl.............................................. -- \7\4.50, -- \10\3.90 -- 4.99 \19\4.49
\9\4.38 ,
\11\3.75
,
\12\4.59
,
\13\4.54
1,2,3,5-Tetrachlorobenzene.................................... -- \7\4.65 4.46 -- 4.99 -- \17\4.70
2,2'-Dichlorobiphenyl......................................... -- \9\4.90 -- \9\4.90, -- 4.65 \20\4.80
\10\3.63
,
\11\3.55
,
\14\4.51
,
\15\5.02
Pentachlorobenzene............................................ -- \7\5.03 4.94 -- 5.71 -- \24\4.99
2,4,5-Trichlorobiphenyl....................................... -- \7\5.51, -- \10\5.67 -- 5.60 \17\5.70
\9\5.81 ,
\10\5.86
,
\15\5.77
2,3,4,5-Tetrachlorobiphenyl................................... -- \4\6.18, -- -- -- 6.04 \17\5.98
\7\5.72
2,2',4,5,5'-Pentachlorobi-phenyl.............................. 6.11 \9\6.50, -- \13\6.11 -- 6.38 \17\6.31
\7\5.92 ,
\12\6.85
2,2',3,3',6,6'-Hexachloro-biphenyl............................ -- \4\5.76, -- -- -- 6.22 \17\6.36
\7\6.63,
\9\6.81
2,2',3,3',4,4',6-Heptachlorobiphenyl.......................... -- \7\6.68 -- -- -- 7.11 \17\6.90
2,2',3,3',5,5',6,6'-Octachlorobiphenyl........................ -- \7\7.11, -- \12\8.42 -- 7.24 \21\7.16
\9\7.14
2,2',3,3',4, 4',5,6,6'-Nona-chlorobiphenyl.................... -- \4\7.52 -- -- -- 7.74 \17\7.63
2,2',3,3',4, 5,5'6,6'-Nona-chlorobiphenyl..................... -- \7\8.16 -- -- -- 7.71 \17\7.94
Decachlorobiphenyl............................................ -- \7\8.26, -- \12\9.60 -- 8.18 \22\8.21
\9\8.20
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ Hansch and Leo (1979). Shake-flask method in paragraph (e)(8) of this section.
[[Page 295]]
\2\ Banerjee, Yalkowski, and Valvani (1980). Shake-flask method in paragraph (e)(1) of this section.
\3\ Hansch and Leo (1984). Estimates log10Kow using the CLogP3 computer program in paragraph (e)(9) of this section.
\4\ Hawker and Connell (1988). Generator column method and an estimation method correlating log10Kow with the total surface area of the molecule in
paragraph (e)(8) of this section.
\5\ Tewari et al. (1982). Generator column method in paragraph (e)(14) of this section.
\6\ Veith, Austin, and Morris (1979). Reverse-phase HPLC method in paragraph (e)(16) of this section.
\7\ Miller et al. (1984). Generator column method in paragraph (e)(11) of this section.
\8\ Chiou and Schmedding (1982). Shake-flask method in paragraph (e)(4) of this section.
\9\ Woodburn, Doucette, and Andren (1984). Generator column method in paragraph (e)(19) of this section.
\10\ Rapaport and Eisenreich (1984). Reverse-phase HPLC method in paragraph (e)(13) of this section.
\11\ Woodburn (1982). Reverse-phase HPLC method in paragraph (e)(18) of this section.
\12\ Bruggemann, Van der Steen, and Hutzinger (1978). Shake-flask method in paragraph (e)(2) of this section.
\13\ Tulp and Hutzinger (1978). Shake-flask method in paragraph (e)(15) of this section.
\14\ Chiou, Porter, and Schmedding (1983). Shake-flask method in paragraph (e)(5) of this section.
\15\ Bruggemann, Van Der Steen , and Hutzinger (1982). Reverse-phase thin-layer chromatography in paragraph (e)(2) of this section.
\16\ Chiou et al. (1977). Shake-flask method in paragraph (e)(3) of this section.
\17\ Average value using all the data.
\18\ Average value using all the data except the datum point 2.46.
\19\ Average value using all the data except the data points 3.90 and 3.75.
\20\ Average value using all the data except the data points 3.63 and 3.55.
\21\ Average value using all the data except the datum point 8.42.
\22\ Average value using all the data except the datum point 9.60.
\23\ Average value using all the data except the datum point 0.32.
\24\ Average value using all the data excluding the estimated datum point 5.71.
(5) Applicability and specificity. The test guideline is designed to
determine the Kow of solid or liquid organic chemicals in the
range log10Kow 1.0 to <=6.0 (10 to <=10\6\).
(c) Test procedure--(1) Test conditions--(i) Special laboratory
equipment--(A)(1) Generator column. Either of two different methods for
connecting to the generator column shall be used depending on whether
the eluted aqueous phase is analyzed by HPLC (Procedure A, as described
in paragraph (c)(3)(iii) of this section) or by solvent extraction
followed by GC analysis, or any other reliable method of solvent extract
(Procedure B, as described in paragraph (c)(3)(iv) of this section).
(2)(i) The design of the generator column is shown in the following
figure 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.042
(ii) The column consists of a 6 mm (\1/4\ in) O.D. pyrex tube joined
to a short enlarged section of 9 mm pyrex tubing
[[Page 296]]
which in turn is connected to another section of 6 mm (\1/4\ in) O.D.
pyrex tubing. Connections to the inlet teflon tubing (\1/8\ in O.D.) and
to the outlet stainless steel tubing (\1/16\ in O.D.) are made by means
of stainless steel fittings with teflon ferrules. The column is enclosed
in a water jacket for temperature control as shown in the following
figure 2:
Figure 2--Setup Showing Generator Column Enclosed in a Water Jacket and
Overall Arrangement of the Apparatus Used in GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.043
(B) Constant temperature bath with circulation pump-bath and capable
of controlling temperature to 25 0.05 [deg]C.
(Procedures A and B, as described in paragraphs (c)(3)(iii) and
(c)(3)(iv) of this section, respectively).
(C) HPLC equipped with a variable wavelength UV absorption detector
operating at a suitable wavelength and a recording integrator (Procedure
A, as described in paragraph (c)(3)(iii) of this section).
(D) Extractor column--6.6 x 0.6 centimeter (cm) stainless steel tube
with end fittings containing 5 micron frits filled with a superficially
porous phase packing (such as Bondapack C18 Corasil: Waters
Associates) (Procedure A, as described in paragraph (c)(3)(iii) of this
section).
(E) Two 6-port high-pressure rotary switching valves (Procedure A,
as described in paragraph (c)(3)(iii) of this section).
(F) Collection vessel--8 x \3/4\ in section of pyrex tubing with a
flat bottom connected to a short section of \3/8\ in O.D. borosilicate
glass tubing. The collecting vessel is sealed with a \3/8\ in teflon cap
fitting (Procedure B, as described in paragraph (c)(3)(iv) of this
section).
(G) GC, or any other reliable analytic equipment, equipped with a
detector sensitive to the solute of interest (Procedure B, as described
in paragraph (c)(3)(iv) of this section).
(ii) Purity of n-octanol and water. Purified n-octanol, described in
paragraph (c)(2)(i) of this section, and water meeting appropriate
American Society for Testing and Materials Type II standards, or an
equivalent grade, are recommended to minimize the effects of dissolved
salts and other impurities. An ASTM Type II water standard is presented
in the reference listed in paragraph (e)(20) of this section).
(iii) Purity of solvents. It is important that all solvents used in
this method be reagent or HPLC grade and contain no impurities which
could interfere with the determination of the test compound.
(iv) Reference compounds. In order to ensure that the HPLC system is
working properly, at least two of the reference compounds listed in
table 1 in paragraph (b)(4)(ii) of this section should be run. Reference
compounds shall be reagent or HPLC grade to avoid interference by
impurities.
(2) Preparation of reagents and solutions--(i) n-Octanol and water.
Very pure n-octanol can be obtained as follows: Wash pure n-octanol
(minimum 98% pure) sequentially with 0.1N H2SO4, with 0.1N
NaOH, then with distilled water until neutral. Dry the n-octanol with
magnesium sulfate and distill twice in a good distillation column under
reduced pressure [b.p. about 80 [deg]C at 0.27 kPa (2 torr)]. The n-
octanol produced should be at least 99.9% pure. Alternatively, a grade
equivalent to Fisher Scientific Co. No. A-402 ``Certified
[[Page 297]]
Octanol-1'' can be used. Reagent-grade water shall be used throughout
the test procedure, such as ASTM Type II water, or an equivalent grade,
as described in paragraph (c)(1)(ii) of this section.
(ii) Presaturated water. Prepare presaturated water with n-octanol
to minimize the depletion of n-octanol from the column when measuring
the Kowof a test chemical. This is very important when the
test chemical is lipophilic and the log10Kow <=4.
(3) Performance of the test. Initially, an approximately 1.0% (w/w)
solution of the test substance in n-octanol is prepared. Precise
measurement of the solute concentration in this solution is required for
the Kowcalculation. Subsequently, the 1.0% (w/w) solution is
coated on the generator column and using either Procedure A or Procedure
B as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section,
the molar concentration of the test substance in reagent-grade water is
determined.
(i) Test solution. The test solution consists of an approximately
1.0% (w/w) solution of the test substance in n-octanol. A sufficient
quantity (about 10-20 milliliter (mL)) of the test solution should be
prepared to coat the generator column. The solution is prepared by
accurately weighing out, using a tared bottle, quantities of both the
test substance and n-octanol required to make a 1.0% (w/w) solution.
When the weights are measured precisely (to the nearest 0.1 milligram
(mg)), knowing the density of n-octanol (0.827 gram (g)/mL at 25
[deg]C), then the molar concentration of the test substance in the n-
octanol is sufficiently accurate for the purposes of the test procedure.
If desired, however, a separate analytical determination (e.g., by GC,
or any other reliable analytical method) may be used to check the
concentration in the test solution. If storage is required, the test
solution should be kept stoppered to prevent volatilization of the test
chemical.
(ii) Test procedures. Prior to the determination of the
Kow of the test chemical, two procedures shall be followed:
(A) The saturated aqueous solution leaving the generator column
shall be tested for the presence of an emulsion, using a Tyndall
procedure (i.e. light scattering). If colloids are present, they must be
removed prior to injection into the extractor column by lowering the
flow rate of water.
(B) The efficiency of removal of the solute (the test chemical) by
solvent extraction from the extractor column shall be determined and
used in the determination of the Kow of the test chemical.
(iii) Procedure A--HPLC method. (A) Procedure A covers the
determination of the aqueous solubility of compounds which absorb in the
UV. Two reciprocating piston pumps deliver the mobile phase (water or
solvent/water mixture) through two 6-port high-pressure rotary valves
and a 30x0.6 cm C18 analytical column to a UV absorption
detector operating at a suitable wavelength. Chromatogram peaks are
recorded and integrated with a recording integrator. One of the 6-port
valves is the sample injection valve used for injecting samples of
standard solutions of the solute in an appropriate concentration for
determining RFs or standard solutions of basic chromate for determining
the sample-loop volume. The other 6-port valve in the system serves as a
switching valve for the extractor column which is used to remove solute
from the aqueous solutions. The HPLC analytical system is shown
schematically in the following figure 3:
Figure 3--Schematic of HPLC--Generator Column Flow System
[[Page 298]]
[GRAPHIC] [TIFF OMITTED] TR15DE00.044
(B) The general procedure for analyzing the aqueous phase after
equilibration is as follows; a detailed procedure is given in paragraph
(c)(3)(iii)(C)(4) of this section:
(1) Direct the aqueous solution from the generator column to
``Waste'' in figure 3 in paragraph (c)(3)(iii)(A) of this section with
the switching valve in the inject position in order to equilibrate
internal surfaces with the solution, thus insuring that the analyzed
sample would not be depleted by solute adsorption on surfaces upstream
from the valve.
(2) At the same time, water is pumped from the HPLC pumps in order
to displace the solvent from the extractor column.
(3) The switching valve is next changed to the load position to
divert a sample of the solution from the generator column through the
extractor column, and the liquid leaving the extractor column is
collected in a tared weighing bottle. During this extraction step, the
HPLC mobile phase is changed to a solvent/water mixture to condition the
analytical column.
(4) After the desired volume of sample is extracted, the switching
valve is returned to the inject position for elution from the extractor
column and analysis. Assuming that all of the solute was adsorbed by the
extractor column during the extraction step, the chromatographic peak
represents all of the solute in the extracted sample, provided that the
extraction efficiency is 100%. If the extraction efficiency is less than
100%, then the extraction efficiency shall be measured and used to
determine the actual amount of the solute extracted.
(5) The solute concentration in the aqueous phase is calculated from
the peak area, the weight of the extracted liquid collected in the
weighing bottle, the extraction efficiency, and the RF.
(C)(1) Determination of the sample-loop volume. Accurate measurement
of the sample loop may be accomplished by using a spectrophotometric
method such as the one described in the reference listed in paragraph
(e)(6) of this section. For this method, measure absorbance,
Aloop, at 373 nanometers (nm) for at least three solutions,
each of which is prepared by collecting from the sample valve an
appropriate number, n, of loopfuls of an aqueous stock solution of
K2CrO4 (1.3% by weight) and diluting to 50 mL with
0.2% KOH. (For a 20 [micro]L loop, use n = 5; for a 50 [micro]L loop,
use n = 2.) Also measure the absorbance, Astock, of the same
stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop
volume to the nearest 0.1 [micro]L using the relation:
Equation 2:
[GRAPHIC] [TIFF OMITTED] TR15DE00.045
(2) Determination of the RF. (i) For all determinations adjust the
mobile phase solvent/water ratio and flow rate to obtain a reasonable
retention time on the HPLC column. For example, typical concentrations
of organic solvent in the mobile phase range from 50
[[Page 299]]
to 100% while flow rates range from 1 to 3 mL/minutes (min); these
conditions often give a 3 to 5 min retention time.
(ii) Prepare standard solutions of known concentrations of the
solute in a suitable solvent. Concentrations must give a recorder
response within the maximum response of the detector. Inject samples of
each standard solution into the HPLC system using the calibrated sample
loop. Obtain an average peak area from at least three injections of each
standard sample at a set detector absorbance unit full scale (AUFS),
i.e., at the same absorbance scale attenuation setting.
(iii) Calculate the RF from the following equation:
Equation 3:
[GRAPHIC] [TIFF OMITTED] TR15DE00.046
(3) Loading of the generator column. (i) The design of the generator
column was described in paragraph (c)(1)(i) of this section and is shown
in figure 1 in paragraph (c)(1)(i)(A)(2)(i) of this section. To pack the
column, a plug of silanized glass wool is inserted into one end of the 6
mm pyrex tubing. Silanized diatomaceous silica support (about 0.5g of
100-120 mesh Chromosorb W chromatographic support material) is poured
into the tube with tapping and retained with a second plug of silanized
glass wool.
(ii) The column is loaded by pulling the test solution through the
dry support with gentle suction and then allowing the excess solution to
drain out. After loading the column, draw water up through the column to
remove any entrapped air.
(4) Analysis of the solute. Use the following procedure to collect
and analyze the solute:
(i) With the switching valve in figure 3 in paragraph (c)(3)(iii)(A)
of this section in the inject position (i.e., water to waste), pump
water through the generator column at a flow rate of approximately 1 mL/
min for approximately 15 min to bring the system into equilibrium. Pump
water to the generator column by means of a minipump or pressurized
water reservoir as shown in the following figure 4:
Figure 4--Water Reservoir for GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.047
(ii) Flush out the organic solvent that remains in the system from
previous runs by changing the mobile phase to 100% H2O and
allowing the water to reach the HPLC detector, as indicated by a
negative reading. As soon as this occurs, place a 25 mL weighing bottle
(weighed to the nearest mg) at the waste position and immediately turn
the switching valve to the load position.
(iii) Collect an amount of water from the generator column (as
determined
[[Page 300]]
by trial and error) in the weighing bottle, corresponding to the amount
of solute adsorbed by the extractor column that gives a reasonable
detector response. During this extraction step, switch back to the
original HPLC mobile phase composition, i.e., solvent/water mixture, to
condition the HPLC analytical column.
(iv) After the desired volume of sample has been extracted, turn the
switching valve back to the inject position in figure 3 in paragraph
(c)(3)(iii)(A) of this section. As soon as the switching valve is turned
to the inject position, remove the weighing bottle, cap it and replace
it with the waste container; at the same time turn on the recording
integrator. The solvent/water mobile phase will elute the solute from
the extractor column and transfer the solute to the HPLC analytical
column.
(v) Determine the weight of water collected to the nearest mg and
record the corresponding peak area. Using the same AUFS setting repeat
the analysis of the solute at least two more times and determine the
average ratio of peak area to grams of water collected. In this
equation, S = solubility (M), RF = response factor, Vloop =
sample-loop volume (L), and R = ratio of area to grams of water.
Calculate the solute solubility in water using the following equation:
Equation 4:
[GRAPHIC] [TIFF OMITTED] TR15DE00.048
(iv) Procedure B--GC Method. In the GC method, or any other reliable
quantitative method, aqueous solutions from the generator column enter a
collecting vessel in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this
section containing a known weight of extracting solvent which is
immiscible in water. The outlet of the generator column is positioned
such that the aqueous phase always enters below the extracting solvent.
After the aqueous phase is collected, the collecting vessel is stoppered
and the quantity of aqueous phase is determined by weighing. The solvent
and the aqueous phase are equilibrated by slowly rotating the collecting
vessel. A small amount of the extracting solvent is then removed and
injected into a GC equipped with an appropriate detector. The solute
concentration in the aqueous phase is determined from a calibration
curve constructed using known concentrations of the solute. The
extraction efficiency of the solvent shall be determined in a separate
set of experiments.
(A) Determination of calibration curve. (1) Prepare solute standard
solutions of concentrations covering the expected range of the solute
solubility. Select a column and optimum GC operating conditions for
resolution between the solute and solvent and the solute and extracting
solvent. Inject a known volume of each standard solution into the
injection port of the GC. For each standard solution determine the
average of the ratio R of peak area to volume (in [micro]L) for the
chromatographic peak of interest from at least three separate
injections.
(2) After running all the standard solutions, determine the
coefficients, a and b, using linear regression analysis on the equation
of concentration (C) vs. R in the form:
Equation 5:
[GRAPHIC] [TIFF OMITTED] TR15DE00.049
(B) Loading of the generator column. The generator column is packed
and loaded with solute in the same manner as for the HPLC method in
paragraph (c)(3)(iii) of this section. As shown in figure 2 in paragraph
(c)(1)(i)(A)(2)(ii) of this section, attach approximately 20 cm of
straight stainless steel tubing to the bottom of the generator column.
Connect the top of the generator column to a water reservoir in figure 4
in paragraph (c)(3)(iii)(C)(4)(i) of this section using teflon tubing.
Use air or nitrogen pressure (5 PSI) from an air or nitrogen cylinder to
force water from the reservoir through the column. Collect water in an
Erlenmeyer flask for approximately 15 min while the solute concentration
in water equilibrates; longer time may be required for less soluble
compounds.
(C) Collection and extraction of the solute. During the
equilibration time, add a known weight of extracting solvent to a
collection vessel which can be capped. The extracting solvent should
[[Page 301]]
cover the bottom of the collection vessel to a depth sufficient to
submerge the collecting tube but still maintain 100:1 water/solvent
ratio. Record the weight (to the nearest mg) of a collection vessel with
cap and extracting solvent. Place the collection vessel under the
generator column so that water from the collecting tube enters below the
level of the extracting solvent in figure 2 in paragraph
(c)(1)(i)(A)(2)(ii) of this section. When the collection vessel is
filled, remove it from under the generator column, replace cap, and
weigh the filled vessel. Determine the weight of water collected. Before
analyzing for the solute, gently rotate the collection vessel contents
for approximately 30 min, controlling the rate of rotation so as not to
form an emulsion; rotating the flask end over end five times per minute
is sufficient. The extraction efficiency of the solvent shall be
determined in a separate set of experiments.
(D) Analysis of the solute. (1) After rotating, allow the collection
vessel to stand for approximately 30 min; then remove a known volume of
the extracting solvent from the vessel using a microliter syringe and
inject it into the GC. Record the ratio of peak area to volume injected
and, from the regression equation of the calibration line, determine the
concentration of solute in the extracting solvent. If the extraction
efficiency is not 100%, the measured extraction efficiency shall be used
to obtain the correct concentration of solute extracted. In this
equation, Ces is the molar concentration of solute in
extracting solvent, dH2O and des are
the densities in grams per milliliter of water and extracting solvent,
respectively, and ges and gH2O are the
grams of extracting solvent and water, respectively, contained in the
collection vessels. The molar concentration of solute in water C(M) is
determined from the following equation:
Equation 6:
[GRAPHIC] [TIFF OMITTED] TR15DE00.050
(2) Make replicate injections from each collecting vessel to
determine the average solute concentration in water for each vessel. To
make sure the generator column has reached equilibrium, run at least two
additional (for a total of three) collection vessels and analyze the
extracted solute as described in paragraph (c)(3)(iv)(D)(1) of this
section. Calculate C(M) from the average solute concentration in the
three vessels.
(3) If another analytical method is used in place of the GC, then
Procedure B, as described in paragraph (c)(3)(iv) of this section, shall
be modified and the new analytical procedure shall be used to determine
quantitatively the amount of solute extracted in the extraction solvent.
(v) Analysis of reference compounds. Prior to analyzing the test
solution, make duplicate runs on at least two of the reference compounds
listed in table 1 in paragraph (b)(4)(ii) of this section. When using
the reference compounds, follow the same procedure previously described
for preparing the test solution and running the test. If the average
value obtained for each compound is within 0.1 log unit of the reference
value, then the test procedure and HPLC system are functioning properly;
if not a thorough checking over of the HPLC and careful adherence to the
test procedures should be done to correct the discrepancy.
(vi) Modification of procedures for potential problems--
Decomposition of the test compound. If the test compound decomposes in
one or more of the aqueous solvents required during the period of the
test at a rate such that an accurate value for water solubility cannot
be obtained, then it will be necessary to carry out detailed
transformation studies, such as hydrolysis studies. If decomposition is
due to aqueous photolysis, then it will be necessary to carry out the
studies in the dark, under red or yellow lights, or by any other
suitable method to eliminate this transformation process.
(d) Data and reporting--(1) Test report. (i) For the test solution,
report the weights to the nearest 0.1 mg of the test substance and n-
octanol. Also report the weight percent and molar concentration of the
test substance in the n-octanol; the density of n-octanol at 25 [deg]C
is 0.827 grams per milliliter (gm)/mL.
(ii) For each run provide the molar concentration of the test
substance in
[[Page 302]]
water for each of three determinations, the mean value, and the standard
deviation.
(iii) For each of the three determinations calculate the
Kow as the ratio of the molar concentration of the test
substance in n-octanol to the molar concentration in water. Also
calculate and report the mean Kow and its standard deviation.
Values of Kow shall be reported as their logarithms
(log10Kow).
(iv) Report the temperature (0.05 [deg]C) at
which the generator column was controlled during the test.
(v) For each reference compound report the individual values of
log10Kow and the average of the two runs.
(vi) For compounds that decompose at a rate such that a precise
value for the solubility cannot be obtained, provide a statement to that
effect.
(2) Specific analytical, calibration, and recovery procedures. (i)
For the HPLC method describe and/or report:
(A) The method used to determine the sample-loop volume and the
average and standard deviation of that volume.
(B) The average and standard deviation of the RF.
(C) The extraction solvent and the extraction efficiency used.
(D) Any changes made or problems encountered in the test procedures.
(ii) For the GC method report:
(A) The column and GC operating conditions of temperature and flow
rate.
(B) The average and standard deviation of the average area per
microliter obtained for each of the standard solutions.
(C) The form of the regression equation obtained in the calibration
procedure.
(D) The extracting solvent and extraction efficiency used.
(E) The average and standard deviation of solute concentration in
each collection vessel.
(F) Any changes made or problems encountered in the test procedure.
(iii) If another approved analytical method is used to determine the
concentration of the test chemical in water, then all the important test
conditions shall be reported.
(iv) If the concentration of the test substance in n-octanol is
determined by an independent analytical method such as GC, provide a
complete description of the method.
(e) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available from the TSCA Nonconfidential Information
Center, Rm. NE-B607, Environmental Protection Agency, 401 M St., SW.,
Washington, DC, 12 noon to 4 p.m., Monday through Friday, excluding
legal holidays.
(1) Banerjee, S. et al., Water solubility and octanol/water
partition coefficient of organics. Limitation of the solubility-
partition coefficient correlation. Environmental Science and Technology
14:1227-1229 (1980).
(2) Bruggemann W.A. et al., Reversed-phase thin-layer chromatography
of polynuclear aromatic hydrocarbons and chlorinated biphenyls.
Relationship with hydrophobicity as measured by aqueous solubility and
octanol/water partition coefficient. Journal of Chromatography 238: 335-
346 (1982).
(3) Chiou, C.T. et al. Partition coefficient and bioaccumulation of
selected organic chemicals. Environmental Science and Technology 11:475-
478 (1977).
(4) Chiou, C.T. and Schmedding, D.W., Partitioning of organic
compounds in octanol/water systems. Environmental Science and Technology
16:4-10 (1982).
(5) Chiou, C.T et al., Partition equilibria of nonionic organic
compounds between soil, organic matter, and water. Environmental Science
and Technology 17:227-231 (1983).
(6) DeVoe, H. et al. ``Generator Columns and High Pressure Liquid
Chromatography for Determining Aqueous Solubilities and Octanol-Water
Partition Coefficients of Hydrophobic Substances,'' Journal of Research
of the National Bureau of Standards, 86:361-366 (1981).
(7) Fujita, T. et al. ``A New Substituent Constant, Derived from
Partition Coefficients.'' Journal of the American Chemical Society,
86:5175 (1964).
(8) Hansch, C. and Leo, A. 1985 MEDCHEM Project, version 26. Pomona
College, Claremont, CA. USA.
[[Page 303]]
(9) Hansch, C. and Leo, A. Medchem Software Manual. CLOGP3 Users
Guide. Release 3.32. December 1984. Medicinal Chemistry Project, Pomona
College, Claremont, CA.
(10) Hawker, D.W. and Connell, D.W. Octanol-water partition
coefficients of polychlorinated biphenyl congeners. Environmental
Science and Technology 22:382-387 (1988).
(11) May, W.E. et al. ``Determination of the aqueous solubility of
polynuclear aromatic hydrocarbons by a coupled column liquid
chromatographic technique,'' Analytical Chemistry, 50:175-179 (1978).
(12) May, W.E. et al. ``Determination of the Solubility Behavior of
Some Polycyclic Aromatic Hydrocarbons in Water,'' Analytical Chemistry
50:997-1000 (1978).
(13) Miller, M.M. et al. Aqueous solubilities, octanol/water
partition coefficients and entropies of melting of chlorinated benzenes
and biphenyls. Journal of Chemical and Engineering Data 29:184-190
(1984).
(14) Neely, W.B. et al. Partition Coefficient to Measure
Bioconcentration Potential of Organic Chemicals in Fish, Environmental
Science Technology, 8:113-115 (1974).
(15) Rappaport, R.A. and Eisenrich, S.J. Chromatographic
determination of octanol-water partition coefficients (Kow's)
for 58 polychlorinated biphenyl congeners. Environmental Science and
Technology 18:163-170 (1984).
(16) Tewari, Y.B. et al. Aqueous solubility and octanol/water
partition coefficients of organic compounds at 25 [deg]C. Journal of
Chemical and Engineering Data 27:451-454 (1982).
(17) Tulp, M.T.M. and Hutzinger, O. Some thoughts on aqueous
solubilities and partition coefficients of PCB, and the mathematical
correlation between bioaccumulation and physio-chemical properties.
Chemosphere 10:849-860 (1978).
(18) Veith, G.D. et al. A rapid method for estimating
log10 P for organic chemicals, Water Research 13:43-47
(1979).
(19) Wasik, S.P. et al. Octanol/water partition coefficient and
aqueous solubilities of organic compounds, Report NBSIR 81-2406 (1981).
National Bureau of Standards, U.S. Department of Commerce, Washington,
DC.
(20) Woodburn, K.B. Measurement and application of the octanol/water
partition coefficients for selected polychlorinated biphenyls. Master's
Thesis (1982), University of Wisconsin at Madison, Madison, WI.
(21) Woodburn, K.B. et al. Generator column determination of
octanol/water partition coefficients for selected polychlorinated
biphenyl congeners. Environmental Science and Technology 18:457-459
(1984).
(22) ASTM D 1193-91 (Approved Sep 15, 1991), ``Standard
Specification for Reagent Water.'' American Society for Testing and
Materials (ASTM), 1916 Race St., Philadelphia, PA 19103.