[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: 40CFR799.6784]
[Page 303-311]
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. 799.6784 TSCA water solubility: Column elution method; shake flask
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 830.7840 (March 1998, revised final
guideline). This source is available at the address in paragraph (f) of
this section.
(b) Introductory information--(1) Prerequisites. Suitable analytical
method, structural formula, vapor pressure curve, dissociation constant,
and hydrolysis independence of pH (preliminary test).
(2) Coefficient of variation. The coefficient of variation on the
mean values reported by the participants of the Organization for
Economic Cooperation and Development (OECD) Laboratory Intercomparison
Testing, Part I, 1979, appeared to be dependent on the chemicals tested
and the test temperatures; it ranges from 0.05 to 0.34 for the column
elution method, and from 0.03 to 1.12 for the flask method.
(3) Qualifying statements. (i) The method is not applicable to
volatile substances. Care should be taken that the substances examined
are as pure as possible and stable in water. It must be ascertained that
the identity of the
[[Page 304]]
substance is not changed during the procedure.
(ii) The column elution method is not suitable for volatile
substances. The carrier material used here may not yet be optimal. This
method is intended for material with solubilities below approximately
10-2 gram/Liter (g/L).
(iii) The flask method is intended for materials with solubility
above 10-2 g/L. It is not applicable to volatile substances;
this method may pose difficulties in the case of surface-active
materials.
(c) Method--(1) Introduction, purpose, scope, relevance,
application, and limits of test. (i) A solution is a homogeneous mixture
of different substances in a solvent. The particle sizes of the
dispersed substances are of the same magnitude as molecules and ions;
therefore, the smallest volumes which can be obtained from a solution
are always of uniform composition.
(ii) Solubility in water is a significant parameter because:
(A) The spatial and temporal movement (mobility) of a substance is
largely determined by its solubility in water.
(B) Water soluble substances gain ready access to humans and other
living organisms.
(C) The knowledge of the solubility in water is a prerequisite for
testing biological degradation and bioaccumulation in water and for
other tests.
(iii) No single method is available to cover the whole range of
solubilities in water, from relatively soluble to very low-soluble
chemicals. A general test guideline for the determination of the
solubility in water must include methods which cover the whole range of
water soluble substances. Therefore, this section includes two methods:
(A) One which applies to substances with low solubilities
(<10-2 g/L), referred to as the ``column elution method.''
(B) The other which applies to substances with higher solubilities
(<=10-2 g/L), referred to as the ``flask method.''
(2) Definition. The solubility in water of a substance is specified
by the saturation mass concentration of the substance in water and is a
function of temperature. The solubility in water is specified in units
of weight per volume of solution. The SI-unit is killogram/meter (kg/
m)\3\; g/L may also be used.
(3) Reference substances. The reference substances need not be
employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied. The values presented in table 1 of this
section are not necessarily representative of the results which can be
obtained with this test method as they have been derived from an earlier
version of the test method.
Table 1--Data for Reference Substances
----------------------------------------------------------------------------------------------------------------
T, Mean (milligram No. of
Method [deg]C (mg)/L) Range (mg/L) labs
----------------------------------------------------------------------------------------------------------------
Fluoranthene
Elution method......................... 15 0.275 0.104 to 0.920 6
25 0.373 0.198 to 1.050 7
Hexachlorobenzene
Elution method......................... 15 9.21 x 10-3 2.06 x 10-3 to 2.16 x 10-2 6
25 9.96 x 10-3 1.19 x 10-3 to 2.31 x 10-2 7
[gamma]-Hexachlorocyclohexane
Elution method......................... 15 6.50 4.43 to 10.5 6
25 9.20 6.64 to 14.5 7
2,4-Dichlorophenoxyacetic acid
Flask method........................... 15 0.633 0.380 to 0.764 5
25 0.812 0.655 to 0.927 5
Mercury(II) chloride:
Flask method........................... 15 53.0 47.7 to 56.5 4
25 66.4 58.3 to 70.4 4
4-Nitrophenol:
Flask method........................... 15 9.95 8.88 to 10.9 6
25 14.8 13.8 to 15.9 6
----------------------------------------------------------------------------------------------------------------
[[Page 305]]
(4) Principle of the test methods. The approximate amount of the
sample and the time necessary to achieve the saturation mass
concentration should be determined in a simple preliminary test.
(i) Column elution method. This method is based on the elution of a
test substance with water from a microcolumn which is charged with an
inert carrier material such as glass beads, silica gel, or sand, and an
excess of test substance. The water solubility is determined when the
mass concentration of the eluate is constant. This is shown by a
concentration plateau as a function of time in the following figure 1:
Figure 1--Concentration versus Time of Substance in the Eluate
[GRAPHIC] [TIFF OMITTED] TR15DE00.051
(ii) Flask method. In this method, the substance (solids must be
pulverized) is dissolved in water at a temperature somewhat above the
test temperature. When saturation is achieved, the mixture is cooled and
kept at the test temperature, stirring as long as necessary to reach
equilibrium. Such a procedure is described in the reference listed in
paragraph (f)(2) of this section. Subsequently, the mass concentration
of the substance in the aqueous solution, which must not contain any
undissolved particles, is determined by a suitable analytical method.
(5) Quality criteria--(i) Repeatability. For the column elution
method <30% is acceptable; for the flask method <15% should be observed.
(ii) Sensitivity. This depends upon the method of analysis, but mass
concentration determinations down to at least 10-6 g/L can be
determined.
(iii) Specificity. These methods should only be applied to:
(A) Pure substance.
(B) Substances that are stable in water.
(C) Slightly soluble substances, i.e. <10-2 g/L for the
column elution method.
(D) Organic substances for the column elution method.
(iv) Possibility of standardization. These methods can be
standardized.
(d) Description of the test procedures--(1) Preparations--(i)
Apparatus--(A) Column elution method. (1) The schematic arrangement of
the system is presented in the following figure 2:
[[Page 306]]
Figure 2--Schematic Test Arrangement
[GRAPHIC] [TIFF OMITTED] TR15DE00.052
(2) Although any size is acceptable, provided it meets the criteria
for reproducibility and sensitivity. The column should provide for a
head space of at least five bed-volumes of water and a minimum of five
samples. Alternatively, the size can be reduced if make-up solvent is
employed to replace the initial five bed-volumes removed with
impurities. A suitable microcolumn is shown in the following figure 3:
[[Page 307]]
Figure 3--Microcolumn (all dimensions in millimeters)
[GRAPHIC] [TIFF OMITTED] TR15DE00.053
(3) The column should be connected to a recycling pump capable of
controlling flows of approximately 25 mL/hours (h). The pump is
connected with polytetrafluoroethylene and/or glass connections. The
column and pump, when assembled, should have provision for sampling the
effluent and equilibrating the head space at atmospheric pressure. The
column material is supported with a small (5 millimeter (mm)) plug of
glass wool, which must also serve to filter particles.
(B) Flask method. For the flask method, the following material is
needed:
(1) Normal laboratory glassware and instrumentation.
(2) A device suitable for the agitation of solutions under
controlled constant temperatures.
(3) A centrifuge (preferably thermostatted), if required with
emulsions.
(4) Equipment for analytical determinations.
(2) Reagents. The substance to be tested should be as pure as
possible, particularly in the flask method where purification is not
provided. The carrier material for the column elution
[[Page 308]]
method should be inert. Possible materials which can be employed are
glass beads and silica. A suitable volatile solvent of analytical
reaction quality should be used to apply the test substance to the
carrier material. Double distilled water from glass or quartz apparatus
should be employed as the eluent or solvent. Water directly from an ion
exchanger must not be used.
(3) Test conditions. The test is preferably run at 20 0.5 [deg]C (293 [deg]K). If temperature dependence is
suspected in the solubility (<= 3%/ [deg]C), two other temperatures
should also be used--both differing from each other and the initially
chosen temperature by 10 [deg]C. In this case the temperature control
should be 0.1 [deg]C. One of these additional
temperatures should be below the initial temperature. The chosen
temperature(s) should be kept constant in all parts of the equipment
(including the leveling vessel).
(4) Performance of the tests--(i) Preliminary test. (A) To
approximately 0.1 g of the sample (solid substances must be pulverized)
in a glass-stoppered 10 milliliter (mL) graduated cylinder, increasing
volumes of distilled water at room temperature are added according to
the steps shown in Table 2 of this section:
Table 2--Determination of Solubility
----------------------------------------------------------------------------------------------------------------
Solubility data step 1 step 2 step 3 step 4 step 5 step 6 step 7
----------------------------------------------------------------------------------------------------------------
Total volume H2O added (mL)...................... 0.1 0.5 1 2 10 100 <=100
Approximate solubility (g/L)..................... <=1,000 200 100 50 10 1 <1
----------------------------------------------------------------------------------------------------------------
(B) After each addition of water to give the indicated total volume,
the mixture is shaken vigorously for 10 min and is visually checked for
any undissolved parts of the sample. If, after a total of 10 mL of water
has been added (step 5), the sample or parts of it remain undissolved,
the contents of the measuring cylinder is transferred to a 100 mL
measuring cylinder which is then filled up with water to 100 mL (step 6)
and shaken. At lower solubilities the time required to dissolve a
substance can be considerably long (24 h should be allowed). The
approximate solubility is given in the table under that volume of added
water in which complete dissolution of the sample occurs. If the
substance is still apparently insoluble, further dilution should be
undertaken to ascertain whether the column elution or flask solubility
method should be used.
(ii) Column elution--(A) Apparatus. (1) The equipment is arranged as
shown in figures 2 and 3 in paragraphs (d)(1)(i)(A)(1) and
(d)(1)(i)(A)(2) of this section. Approximately 600 milligrams (mg) of
carrier material is weighed and transferred to a 50 mL round-bottom
flask. A suitable, weighed amount of test substance is dissolved in the
chosen solvent, and an appropriate amount of the test substance solution
is added to the carrier material. The solvent must be completely
evaporated, e.g. in a rotary evaporator; otherwise water saturation of
the carrier is not achieved due to partition effects on the surface of
the carrier.
(2) The loading of carrier material may cause problems (erroneous
results) if the test substance is deposited as an oil or a different
crystal phase. The problem should be examined experimentally.
(3) The loaded carrier material is allowed to soak for about 2 h in
approximately 5 mL of water, and then the suspension is added to the
microcolumn. Alternatively, dry loaded carrier material may be poured in
the microcolumn, which has been filled with water and then equilibrated
for approximately 2 h.
(B) Test procedure. The elution of the substance from the carrier
material can be carried out in two different ways: Leveling vessel or
circulating pump. The two principles should be used alternatively.
(1) Leveling vessel, see figure 3 in paragraph (d)(1)(i)(A)(2) and
figure 4 in paragraph (d)(4)(iii) of this section.
(i) The connection to the leveling vessel is made by using a ground
glass joint which is connected by teflon tubing. It is recommended that
a flow rate
[[Page 309]]
of approximately 25 mL/h be used. Successive eluate fractions should be
collected and analyzed by the chosen method.
(ii) Fractions from the middle eluate range where the concentrations
are constant (30%) in at least five consecutive
fractions are used to determine the solubility in water.
(iii) A second run is to be performed at half the flow rate of the
first. If the results of the two runs are in agreement, the test is
satisfactory; if there is a higher apparent solubility with the lower
flow rate, then the halving of the flow rate must continue until two
successive runs give the same solubility.
(2) Circulating pump, see figures 2 and 3 in paragraphs
(d)(1)(i)(A)(1) and (d)(1)(i)(A)(2) of this section.
(i) With this apparatus, the microcolumn must be modified. A
stopcock with 2-way action must be used, see figure 3 in paragraph
(d)(1)(i)(A)(2) of this section). The circulating pump can be, e.g. a
peristaltic pump (be careful that no contamination and/or adsorption
occurs with the tube material) or a membrane pump.
(ii) The flow through the column is started. It is recommended that
a flow rate of approximately 25 mL/h be used (approximately 10 bed
volumes per h for the described column). The first five-bed volumes
(minimum) are discarded to remove water soluble impurities.
(iii) Following this, the recycling pump is connected and the
apparatus allowed to run until equilibration is established, as defined
by five successive samples whose concentrations do not differ by more
than 30% in a random fashion (see paragraph (f)(2) of this section).
These samples should be separated from each other by time intervals
corresponding to the passage of at least 10 bed-volumes of the eluent.
(3) In both cases (using a circulation pump or a leveling vessel)
the fractions should be checked for the presence of colloidal matter by
examination for the Tyndall effect (light scattering). Presence of such
particles invalidates the results, and the test should be repeated with
improvements in the filtering action of the column. The pH of each
sample should be recorded. A second run should be performed at the same
temperature.
(iii) Flask method: Test procedure. The quantity of material
necessary to saturate the desired volume of water is estimated from the
preliminary test. The volume of water required will depend on the
analytical method and the solubility range. About five times the
quantity of material determined in paragraph (d)(4)(i)(A) of this
section is weighed into each of three glass vessels fitted with glass
stoppers (e.g. centrifuge tubes, flasks). The chosen volume of water is
added to each vessel, and the vessels are tightly stoppered. The closed
vessels are then agitated at 30 [deg]C. (A shaking or stirring device
capable of operating at constant temperature should be used, e.g.
magnetic stirring in a thermostatically controlled water bath.) After 1
day, one of the vessels is removed and re-equilibrated for 24 h at the
test temperature with occasional shaking. The contents of the vessel are
then centrifuged at the test temperature, and the concentration of
compound in the clear aqueous phase is determined by a suitable
analytical method. The other two flasks are treated similarly after
initial equilibration at 30 [deg]C for 2 and 3 days, respectively. If
the concentration results from at least the last two vessels agree with
the required reproducibility, the test is satisfactory. The whole test
should be repeated, using longer equilibration times if the results from
vessels one, two, and three show a tendency to increasing values. The
arrangement of the apparatus is shown in the following figure 4:
[[Page 310]]
Figure 4--Test Arrangement for the Determination of Solubility in Water
of Slightly Soluble, Low Volatility Organic Substances
[GRAPHIC] [TIFF OMITTED] TR15DE00.054
1 = Leveling vessel (e.g. 2.5 L chemical flask)
2 = Column (see figure 3 in paragraph (d)(1)(i)(A)(2) of this section)
3 = Fraction accumulator
4 = Thermostat
5 = Teflon tubing
6 = Glass stopper
7 = Water line (between thermostat and column, inner diameter:
approximately 8 mm)
(iv) Analysis. A substance-specific analytical method is required
for these determinations, since small amounts of soluble impurities can
cause large errors in the measured solubility. Examples of such methods
are gas or liquid chromatography, titration methods, photometric
methods, and polarographic methods.
(e) Data and reporting--(1) Column elution method--(i) Treatment of
results. The mean value from at least five consecutive samples taken
from the saturation plateau (figure 1 in paragraph (c)(4)(i) of this
section) should be determined for each run, as should the standard
deviation. A comparison should be made between the two means to ensure
that they agree with a repeatability of less than 30%.
(ii) Test report. The report should contain an indication of the
results of the preliminary test plus the following information:
(A) The individual concentrations, flow rates and pHs of each
samples.
(B) The means and standard deviations from at least five samples
from the saturation plateau of each run.
(C) The average of the two successive, acceptable runs.
(D) The temperature of the runs.
(E) The method of analysis employed.
(F) The nature of the carrier material employed.
(G) Loading of carrier material.
(H) Solvent used.
[[Page 311]]
(I) Statement that the identity of the substance in the saturated
solution has been proved.
(2) Flask method--(i) Treatment of results. The individual results
should be given for each of the three flasks and those results deemed to
be constant (repeatability <15%) should be averaged and given in units
of mass per volume of solution. This may require the conversion of mass
units to volume units, using the density when the solubility is very
high (100 g/L).
(ii) Test report. The report should include the following
information:
(A) The individual analytical determinations and the average where
more than one value was determined for each flask.
(B) The average of the value for the different flasks which were in
agreement.
(C) The test temperature.
(D) The analytical method employed.
(f) 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) Veith, G.D. and V.M. Comstock. Apparatus for continuously
saturating water with hydrophobic organic chemicals. Journal of the
Fishing Research Board of Canada 32:1849-1851 (1975).
(2) Organization for Economic Cooperation and Development,
Guidelines for The Testing of Chemicals, OECD 105, Water Solubility
(Column Elution Method--Shake Flask Method), OECD, Paris, France (1981).