[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: 40CFR799.6786]
[Page 311-321]
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. 799.6786 TSCA water solubility: 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 Toxics
(OPPTS) harmonized test guideline 830.7860 (March 1998, revised final
guideline). The source is available at the address in paragraph (e) of
this section.
(b) Introduction--(1) Purpose. (i) The water solubility of a
chemical is defined as the equilibrium concentration of the chemical in
a saturated aqueous solution at a given temperature and pressure. The
aqueous phase solubility is an important factor in governing the
movement, distribution, and rate of degradation of chemicals in the
environment. Substances that are relatively water soluble are more
likely to be widely distributed by the hydrologic cycle than those which
are relatively insoluble. Furthermore, substances with higher water
solubility are more likely to undergo microbial or chemical degradation
in the environment because dissolution makes them ``available'' to
interact and, therefore, react with other chemicals and microorganisms.
Both the extent and rate of degradation via hydrolysis, photolysis,
oxidation, reduction, and biodegradation depend on a chemical being
soluble in water (i.e., homogeneous kinetics).
(ii) Water provides the medium in which many organisms live, and
water is a major component of the internal environment of all living
organisms (except for dormant stages of certain life forms). Even
organisms which are adapted to life in a gaseous environment require
water for normal functioning. Water is thus the medium through which
most other chemicals are transported to and into living cells. As a
result, the extent to which chemicals dissolve in water will be a major
determinant for movement through the environment and entry into living
systems.
(iii) The water solubility of a chemical also has an effect on its
sorption into and desorption from soils and sediments, and on
volatilization from aqueous media. The more soluble a chemical substance
is, the less likely it is to sorb to soils and sediments and the less
likely it is to volatilize from water. Finally, the design of most
chemical tests and many ecological and health tests requires precise
knowledge of the water solubility of the chemical to be tested.
(2) Definitions. The following definitions apply to this section.
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Concentration (C) of a solution is the amount of solute in a given
amount of solvent or solution and can be expressed as a weight/weight or
weight/volume relationship. The conversion from a weight relationship to
one of volume incorporates density as a factor. For dilute aqueous
solutions, the density of the solvent is approximately equal to the
density of the solution; thus, concentrations expressed in milligrams
per liter (mg/L) are approximately equal to 10-3 g/10\3\ g or
parts per million (ppm); those expressed in micrograms per liter
([micro]g/L) are approximately equal to 10-6 g/10\3\ g or
parts per billion (ppb). In addition, concentration can be expressed in
terms of molarity, normality, molality, and mole fraction. For example,
to convert from weight/volume to molarity molecular mass is incorporated
as a factor.
Density is the mass of a unit volume of a material. It is a function
of temperature, hence the temperature at which it is measured should be
specified. For a solid, it is the density of the impermeable portion
rather than the bulk density. For solids and liquids, suitable units of
measurement are grams per cubic centimeter (g/cm\3\). The density of a
solution is the mass of a unit volume of the solution and suitable units
of measurement are g/cm\3\.
Extractor column is used to extract the solute from the saturated
solutions produced by the generator column. After extraction onto a
chromatographic support, the solute is eluted with a solvent/water
mixture and subsequently analyzed by high-pressure liquid chromatography
(HPLC), gas chromatography (GC), or any other suitable analytical
procedure. A detailed description of the preparation of the extractor
column is given in paragraph (c)(1)(i)(D) of this section.
Generator column is used to produce or generate saturated solutions
of a solute in a solvent. The column, see figure 1 in paragraph
(c)(1)(i)(A) of this section, is packed with a solid support coated with
the solute, i.e., the organic compound whose solubility is to be
determined. When water (the solvent) is pumped through the column,
saturated solutions of the solute are generated. Preparation of the
generator column is described in paragraph (c)(1)(i)(A) of this section.
Response factor (RF) is the solute concentration required to give a
1 unit area chromatographic peak or 1 unit output from the HPLC
recording integrator at a particular recorder 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)(ii)(B)(2) of this
section.
Sample loop is a \1/16\ inch (in) outer 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)(ii)(B)(1) of this section when the HPLC method is used.
Saturated solution is a solution in which the dissolved solute is in
equilibrium with an excess of undissolved solute; or a solution in
equilibrium such that at a fixed temperature and pressure, the
concentration of the solute in the solution is at its maximum value and
will not change even in the presence of an excess of solute.
Solution is a homogeneous mixture of two or more substances
constituting a single phase.
(3) Principle of the test method. (i) This test method is based on
the dynamic coupled column liquid chromatographic (DCCLC) technique for
determining the aqueous solubility of organic compounds that was
initially developed by May et al. (as described in the references listed
in paragraphs (e)(5) and (e)(6) of this section), modified by DeVoe et
al. (as described in the reference listed in paragraph (e)(1) of this
section), and finalized by Wasik et al. (as described in the reference
listed in paragraph (e)(11) of this section). The DCCLC technique
utilizes a generator column, extractor column and HPLC coupled or
interconnected to provide a continuous closed flow system. Saturated
aqueous solutions of the test compound are produced by pumping water
through the generator column that is packed with a
[[Page 313]]
solid support coated with the compound. The compound is extracted from
the saturated 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) 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, i.e., the water solubility of the
compound, is determined from the mass of the compound (solute) extracted
from a measured volume of water (solvent).
(ii) Since the HPLC method is only applicable to compounds that
absorb in the UV, an alternate GC method, or any other reliable
procedure (which must be approved by OPPTS), can 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. Table 1 of this section lists the water
solubilities at 25 [deg]C for a number of reference chemicals as
obtained from the scientific literature. The data from Wasik et al. (as
described in the reference listed in paragraph (e)(11) of this section),
Miller et al. and Tewari et al. (as described in the references listed
in paragraphs (e)(7) and (e)(10) of this section, respectively) were
obtained from the generator column method. The water solubilities data
were also obtained from Mackay et al. and Yalkowski et al. (as described
in the references listed in paragraphs (e)(4) and (e)(12) of this
section, respectively) and other scientists by the conventional shake
flask method. These data have been provided primarily so that the
generator column method can be calibrated from time to time and to allow
the chemical testing laboratory an opportunity to compare its results
with those listed in table 1 of this section. The water solubility
values at 25 [deg]C reported by Yalkowski et al. are their preferred
values and, in general, represent the best available water solubility
data at 25 [deg]C. The testing laboratory has the option of choosing its
own reference chemicals, but references must be given to establish the
validity of the measured values of the water solubility.
Table 1--Water Solubilities at 25 [deg]C of Some Reference Chemicals
----------------------------------------------------------------------------------------------------------------
Water solubility (ppm at 25 [deg]C)
--------------------------------------------------------
Reference chemical Wasik (generator Other literature
column method) Yalkowski\1\ \5\ references
----------------------------------------------------------------------------------------------------------------
2-Heptanone............................................ \2\4080 4300 \5\4330
1-Chlorobutane......................................... \2\873 872.9 \7\666
Ethylbenzene........................................... \2\187 208 \7\162
1,2,3-Trimethylbenzene................................. \2\65.5 75.2 \7\48.2
Biphenyl............................................... \3\ \10\6.71 7.48 \8\6.62
Phenanthrene........................................... \4\1.002 1.212 --
2,4,6-Trichlorobiphenyl................................ \3\ \10\0.226 0.225 \8\0.119
2,3,4,5-Tetrachlorobiphenyl............................ \3\ \10\0.0209 0.01396 \8\0.0192
Hexachlorobenzene...................................... -- 0.004669 \9\0.00996
2,3,4,5,6-Pentachlorobiphenyl.......................... \3\ \10\0.00548 0.004016 \8\0.0068
----------------------------------------------------------------------------------------------------------------
\1\ Preferred water solubility at 25 [deg]C by Yalkowski et al. (1990) in paragraph (e)(12) of this section
based on a critical review of all the experimental water solubility data published.
\2\ Tewari et al. (1982) in paragraph (e)(10) of this section.
\3\ Leifer et al. (1983) in paragraph (e)(3) of this section.
\4\ May, Wasik, and Freeman (1978, 1978a) in paragraphs (e)(5) and (6) of this section.
\5\ Yalkowski et al. (1990) in paragraph (e)(12) of this section.
\6\ Hansch et al. (1968) in paragraph (e)(2) of this section.
\7\ Sutton and Calder (1975) in paragraph (e)(9) of this section.
\8\ Mackay et al. (1980) in paragraph (e)(4) of this section.
\9\ The elution chromatographic method from Organization for Economic Cooperation and Development (OECD) (1981)
in paragraph (e)(8) of this section.
\10\ Miller et al. (1984) in paragraph (e)(7) of this section.
[[Page 314]]
(5) Applicability and specificity. (i) Procedures are described in
this section to determine the water solubility for liquid or solid
compounds. The water solubility can be determined in very pure water,
buffer solution for compounds that reversibly ionize or protonate, or in
artificial seawater as a function of temperature (i.e., in the range of
temperatures of environmental concern). This section is not applicable
to the water solubility of gases.
(ii) This section is designed to determine the water solubility of a
solid or liquid test chemical in the range of 1 ppb to 5,000 ppm. For
chemicals whose solubility is below 1 ppb, the water solubility should
be characterized as ``less than 1 ppb'' with no further quantification.
For solubilities greater than 5,000 ppm, the shake flask method should
be used, see paragraph (e)(15) of this section.
(c) Test procedure--(1) Test conditions--(i) Special laboratory
equipment--(A) Generator column. (1) Either of two different designs
shall be used depending on whether the eluted aqueous phase is analyzed
by HPLC in paragraph (c)(3)(ii) of this section or by solvent extraction
followed by GC (or any other reliable quantitative) analysis of solvent
extract in paragraph (c)(3)(iv) of this section. The design of the
generator column is shown in the following figure 1:
Figure 1--Generator Column
[GRAPHIC] [TIFF OMITTED] TR15DE00.055
(2) 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 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.) shall be made by means of
stainless steel fittings with teflon ferrules. The column
[[Page 315]]
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 the GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.056
(B) Constant temperature bath with circulation pump-bath and capable
of controlling temperature to 0.05 [deg]C, see
paragraph (c)(3) of this section.
(C) HPLC equipped with a variable wavelenth UV absorption detector
operating at a suitable wavelength and a recording integrator in
paragraph (c)(3)(ii) of this section.
(D) Extractor column--6.6 x 0.6 cm stainless steel tube with end
fittings containing 5 [micro]m frits filled with a superficially porous
phase packing (Bondapack C18/Corasil: Waters Associates) in paragraph
(c)(3)(ii) of this section.
(E) Two 6-port high-pressure rotary switching valves in paragraph
(c)(3)(ii) 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 in figure 2 in paragraph (c)(1)(i)(A)(2) of this section.
The collecting vessel is sealed with a \3/8\ in teflon cap fitting in
paragraph (c)(3)(iii) of this section.
(G) GC, or any other reliable analytical equipment, which has a
detector sensitive to the solute of interest in paragraph (c)(3)(iii) of
this section.
(ii) Purity of water. Water meeting appropriate American Society for
Testing and Materials (ASTM) Type II standards, or an equivalent grade,
are recommended to minimize the effects of dissolved salts and other
impurities on water solubility. ASTM Type II water is presented in the
reference listed in paragraph (e)(13) of this section.
(iii) Purity of solvents. All solvents used in this method must be
reagent or HPLC grade. Solvents must contain no impurities which could
interfere with the determination of the test compound.
(iv) Seawater. When the water solubility in seawater is desired, the
artificial seawater described in paragraph (c)(2)(ii) of this section
must be used.
(v) Effect of pH on solubility. For chemicals that reversibly ionize
or protonate with a pKa or pKb between 3 and 11,
experiments must be performed at pH's 5.0, 7.0, and 9.0 using
appropriate buffers.
(2) Preparation of reagents and solutions--(i) Buffer solutions.
Prepare buffer solutions as follows:
(A) pH 3.0--to 250 mL of 0.10M potassium hydrogen phosphate add 111
mL of 0.10 M hydrochloric acid; adjust the final volume to 500 mL with
reagent grade water.
(B) pH 5.0--to 250 mL of 0.1M potassium hydrogen phthalate add 113
mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with
reagent grade water.
(C) pH 7.0--to 250 mL of 0.1M potassium dihydrogen phosphate add 145
mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with
reagent grade water.
(D) pH 9.0--to 250 mL of 0.075M borax add 69 mL of 0.1M HCl; adjust
the final volume to 500 mL with reagent grade water.
(E) pH 11.0--to 250 mL of 0.05 M sodium bicarbonate add 3 mL of 0.10
M
[[Page 316]]
sodium hydroxide; adjust the final volume to 500 mL with reagent grade
water.
(ii) Check the pH of each buffer solution with a pH meter at 25
[deg]C and adjust to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the
solution has changed by 0.2 pH units or more after
the addition of the test compound, then a more concentrated buffer is
required for that pH determination. The sponsor should then choose a
more suitable buffer.
(iii) Artificial seawater. Add the reagent-grade chemicals listed in
table 2 of this section in the specified amounts and order to 890 mL of
reagent-grade water. Each chemical shall be dissolved before another one
is added.
Table 2--Constituents of Artificial Seawater\1\
------------------------------------------------------------------------
Chemical Amount
------------------------------------------------------------------------
NaF.................................................. 3 mg
SrCl2.6H2O........................................... 20 mg
H3BO3................................................ 30 mg
KBr.................................................. 100 mg
KCl.................................................. 700 mg
CaCl2.2H2O........................................... 1.47 gram (g)
Na2SO4............................................... 4.00 g
MgCl2.6H2O........................................... 10.78 g
NaCl................................................. 23.50 g
Na2SiO3.9H2O......................................... 20 mg
NaHCO3............................................... 200 mg
------------------------------------------------------------------------
\1\ If the resulting solution is diluted to 1 L, the salinity should be
34 0.5 g/kilogram (kg) and the pH 8.0 0.2. The desired test salinity is attained by dilution
at time of use.
(3) Performance of the test. Using either the procedures in
paragraph (c)(3)(ii) or (c)(3)(iii) of this section, determine the water
solubility of the test compound at 25 [deg]C in reagent-grade water or
buffer solution, as appropriate. Under certain circumstances, it may be
necessary to determine the water solubility of a test compound at 25
[deg]C in artificial seawater. The water solubility can also be
determined at other temperatures of environmental concern by adjusting
the temperature of the water bath to the appropriate temperature.
(i) Prior to the determination of the water solubility of the test
chemical, two procedures shall be followed.
(A) The saturated aqueous solution leaving the generator column must
be tested for the presence of an emulsion, using a Tyndall procedure. If
colloids are present, they must be eliminated prior to the injection
into the extractor column. This may be achieved by lowering the flow
rate of the water.
(B) The efficiency of the removal of the solute (i.e. test chemical)
by the solvent extraction from the extraction column must be determined
and used in the determination of the water solubility of the test
chemical.
(ii) Procedure A--HPLC method--(A) Scope. (1) Procedure A covers the
determination of the aqueous solubility of compounds which absorb in the
UV.
(i) The HPLC analytical system is shown schematically in the
following figure 3:
Figure 3--Schematic of HPLC--Generator Column Flow System
[GRAPHIC] [TIFF OMITTED] TR15DE00.057
[[Page 317]]
(ii) Two reciprocating piston pumps deliver the mobile phase (water
or solvent/water mixture) through two 6-port high-pressure rotary valves
and a 30 x 0.6 cm C18/Corasil analytical column to a variable wavelength
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 of 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.
(2) The general procedure for analyzing the aqueous phase is as
follows (a detailed procedure is given in paragraph (c)(3)(ii)(B)(4) of
this section).
(i) Direct the aqueous solution to ``Waste,'' see figure 3 in
paragraph (c)(3)(ii)(A)(1)(i) of this section, with the switching valve
in the inject position in order to equilibrate internal surfaces with
the solution, thus ensuring that the analyzed sample would not be
depleted by solute adsorption on surfaces upstream from the valve.
(ii) At the same time, water is pumped from the HPLC pumps in order
to displace the solvent from the extractor column.
(iii) The switching valve is next changed to the load position to
divert a sample of the solution through the extractor column, and the
liquid leaving this column is collected in a weighing bottle. During
this extraction step, the mobile phase is changed to a solvent/water
mixture to condition the analytical column.
(iv) After the desired volume of sample is extracted, the switching
valve is returned to the inject position for elution and analysis.
Assuming that there is no breakthrough of solute from the extractor
column during the extraction step, the chromatographic peak represents
all of the solute in the sample, provided that the extraction efficiency
is 100%. If the extraction efficiency is less than 100%, then the
extraction efficiency shall be used to determine the actual weight of
the solute extracted.
(v) The solute concentration in the aqueous phase is calculated from
the peak area and the weight of the extracted liquid collected in the
weighing bottle.
(B) Determinations--(1) Sample-loop volume. Accurate measurement of
the sample loop may be accomplished by using the spectrophotometric
method of Devoe et al. under paragraph (e)(1) of this section. For this
method measure absorbance, Aloop, at 373 nm of 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 equation:
Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.059
(2) 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 solvent in the
mobile phase range from 50 to 100% while flow rates range from 1 to 3
mL/min; these conditions 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 absorbance unit full scale (AUFS), i.e., at the
same absorbance scale attenuation setting.
(iii) Calculate the RF from the following equation:
Equation 2:
[[Page 318]]
[GRAPHIC] [TIFF OMITTED] TR15DE00.058
(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) 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 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) If the solute is a liquid, the column is loaded by pulling the
liquid solute through the dry support with gentle suction. If the solute
is a solid, a 1% solution of the solid in a volatile solvent is added to
the dry packing. The solvent is then distilled off the column under
reduced pressure. After loading the column draw water up through the
column to remove entrapped air.
(4) Analysis of the solute. Use the following procedure to collect
and analyze the solute.
(i) With the switching valve (figure 3 in paragraph
(c)(3)(ii)(A)(1)(i) 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 5 minutes (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] TR15DE00.060
(ii) Flush out the 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 (as determined by trial and error)
in the weighing bottle, corresponding to the amount of solute adsorbed
by the extractor column that gives a large on-scale 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
[[Page 319]]
switching valve back to the inject position (figure 3 in paragraph
(c)(3)(ii)(A)(1)(i) of this section); 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) Remove the weighing bottle, cap it, and replace it with the
waste container. 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 3:
[GRAPHIC] [TIFF OMITTED] TR15DE00.061
(iii) Procedure B--GC method--(A) Scope. In the GC method, or any
other analytical method, aqueous solutions from the generator column
enter a collecting vessel (figure 2 in paragraph (c)(1)(i)(A)(2) 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. The extraction efficiency of the solvent must be determined at
this time. A small amount of the extracting solvent is removed and
injected into a gas chromograph 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.
(B) Alternative method. If another (approved) analytical method is
used instead of the GC, that method shall be used to determine
quantitatively the amount of solute present in the extraction solvent.
(C) Determinations--(1) Calibration curve. (i) Prepare solute
standard solutions of concentrations covering the 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 microliters) for three
chromatographic peaks from three injections.
(ii) After running all the standard solutions, determine the
coefficients, a and b, using a linear regression equation of C vs. R in
the following form:
Equation 4:
[GRAPHIC] [TIFF OMITTED] TR15DE00.062
(iii) If another analytical method is used, the procedures described
in paragraph (c)(3)(iii)(C)(1) of this section shall be used to
determine quantitatively the amount of solute in the extraction solvent.
(2) Loading of the generator column. The generator column is packed
and loaded with solute in the same manner as for the HPLC method
described under paragraph (c)(3)(ii)(B)(3) of this section. As shown in
figure 2 in paragraph (c)(1)(i)(A)(2) 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 (figure 4 in paragraph (c)(3)(ii)(B)(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.
(3) 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 320]]
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 (figure 2 in paragraph (c)(1)(i)(A)(2)
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 shake the collection vessel contents for approximately 30
min, controlling the rate of shaking so as not to form an emulsion;
rotating the flask end over end five times per minute is sufficient.
(4) Analysis of the solute. (i) After shaking, 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. In this equation,
Ces is the concentration of solute in extracting solvent (M),
dH2O and des are the densities of water and
extracting solvent, respectively, and ges and gH2O
are the grams of extracting solvent and water, respectively, contained
in the collection vessel. The concentration of solute in water C(M) is
determined from the following equation:
Equation 5:
[GRAPHIC] [TIFF OMITTED] TR15DE00.063
(ii) 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 above. Calculate the water solubility of
the solute from the average solute concentration in the three vessels.
(iv) Modification of procedures for potential problems. 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; e.g., hydrolysis in paragraph
(e)(16) of this section. If decomposition is due to aqueous photolysis,
then it will be necessary to carry out water solubility 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 each set of
conditions, (e.g., temperature, pure water, buffer solution, artificial
seawater) required for the study, provide the water solubility value for
each of three determinations, the mean value, and the standard
deviation.
(ii) For compounds that decompose at a rate such that a precise
value for the water solubility cannot be obtained, provide a statement
to that effect.
(iii) For compounds with water solubility below 1 ppb, report the
value as ``less than 1 ppb.''
(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) Any changes made or problems encountered in the test procedure.
(ii) For the GC, or any other analytical, method report:
(A) The column and GC operating conditions of temperature and flow
rate, or the operating conditions of any other analytical method used.
(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.
[[Page 321]]
(D) The extracting solvent used, and its extraction efficiency.
(E) The average and standard deviation of solute concentration in
each collection vessel.
(F) Any changes made or problems encountered in the test procedure.
(G) If applicable, a complete description of the analytical method
which was used instead of the GC method.
(e) References. For additional 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) 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,
National Bureau of Standards, 86:361-366 (1981).
(2) Hansch, C. et al., The linear free-energy relationship between
partition coefficients, and the aqueous solubility of organic liquids.
Journal of Organic Chemistry 33:347-350 (1968).
(3) Leifer, A. et al., Environmental transport and transformation of
polychlorinated biphenyls. Chapter 1. U.S. Environmental Protection
Agency Report: EPA-560/5-83-005 (1983).
(4) Mackay, D. et al., Relationships between aqueous solubility and
octanol-water partition coefficient. Chemosphere 9:701-711 (1980).
(5) 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).
(6) May, W.E. et al. Determination of the solubility behavior of
some polycyclic aromatic hydrocarbons in the water. Analytical
Chemistry, 50:997-1000 (1978a).
(7) Miller, N.M. et al., Aqueous solubilities, octanol/water
partition coefficients, and entropy of melting of chlorinated benzenes
and biphenyls. Journal of Chemical and Engineering Data 29:184-190
(1984).
(8) OECD/Organization for Economic Cooperation and Development. Test
Guideline No. 105. Water solubility column elution-flask method (1981).
(9) Sutton, C. and Calder, J.A., Solubility of alkylbenzenes in
distilled water and seawater at 25 [deg]C. Journal of Chemical and
Engineering Data 20:320-322 (1975).
(10) Tewari, Y.B. et al., Aqueous solubility and octanol/water
partition coefficient of organic compounds at 25 [deg]C. Journal of
Chemical and Engineering Data 27:451-454 (1982).
(11) Wasik, S.P. et al., Octanol/Water Partition Coefficient and
Aqueous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406.
Washington, DC: National Bureau of Standards, U.S. Department of
Commerce (1981).
(12) Yalkowski, S.H. et al., ``Aquasol database of aqueous
solubilities of organic compounds''; Fifth Edition. University of
Arizona, College of Pharmacy, Tucson, AZ 85721 (1990) (available at
http://www.pharm.arizona.edu/aquasol/index.html).
(13) ASTM D 1193-91, Standard Specification for Reagent Water.
American Society for Testing and Materials (ASTM). 1916 Race St.,
Philadelphia, PA 19103.
Subparts F-G [Reserved]