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§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 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
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T, Mean (milligram No. of
Method °C (mg)/L) Range (mg/L) labs
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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[SU]-3[/ 2.06 x 10[SU]-3[/SU] to 2.16 6
SU] x 10[SU]-2[/SU]
25 9.96 x 10[SU]-3[/ 1.19 x 10[SU]-3[/SU] to 2.31 7
SU] x 10[SU]-2[/SU]
[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
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(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:
(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:
(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:
(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 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 °C (293 °K).
If temperature dependence is suspected in the solubility (≤ 3%/ °C), two other
temperatures should also be used -- both differing from each other and the
initially chosen temperature by 10 °C. In this case the temperature control
should be ±0.1 °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: (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 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 °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 °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:
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.
(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).
Table 2_Determination of Solubility
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Solubility data step 1 step 2 step 3 step 4 step 5 step 6 step 7
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Total volume H[INF]2[/INF]O added (mL)........... 0.1 0.5 1 2 10 100 [le]100
Approximate solubility (g/L)..................... [le]1,00 200 100 50 10 1 <1
0
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