[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: 40CFR795.70]
[Page 46-59]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 795_PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart B_Provisional Chemical Fate Guidelines
Sec. 795.70 Indirect photolysis screening test: Sunlight photolysis in
waters containing dissolved humic substances.
Subpart A [Reserved]
Subpart B_Provisional Chemical Fate Guidelines
Sec.
795.70 Indirect photolysis screening test: Sunlight photolysis in waters
containing dissolved humic substances.
Subpart C_Provisional Environmental Effects Guidelines
795.120 Gammarid acute toxicity test.
Subpart D_Provisional Health Effects Guidelines
795.225 Dermal pharmacokinetics of DGBE and DGBA.
795.228 Oral/dermal pharmacokinetics.
795.231 Pharmacokinetics of isopropanal.
795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
795.250 Developmental neurotoxicity screen.
Authority: 15 U.S.C. 2603.
Subpart A [Reserved]
(a) Introduction. (1) Chemicals dissolved in natural waters are
subject to two types of photoreaction. In the first case, the chemical
of interest absorbs sunlight directly and is transformed to products
when unstable excited states of the molecule decompose. In the second
case, reaction of dissolved chemical is the result of chemical or
electronic excitation transfer from light-absorbing humic species in the
natural water. In contrast to direct photolysis, this photoreaction is
governed initially by the spectroscopic properties of the natural water.
(2) In general, both indirect and direct processes can proceed
simultaneously. Under favorable conditions the measurement of a
photoreaction rate constant in sunlight (KpE) in a natural
water body will yield a net value that is the sum of two first-order
reaction rate constants for the direct (kDE) and indirect
(kIE) pathways which can be expressed by the relationship
Equation 1
kpE=kDE+kIE.
This relationship is obtained when the reaction volume is optically thin
so that a negligible fraction of the incident light is absorbed and is
sufficiently dilute in test chemical; thus the direct and indirect
photoreaction processes become first-order.
(3) In pure water only, direct photoreaction is possible, although
hydrolysis, biotransformation, sorption, and volatilization also can
decrease the concentraton of a test chemical. By measuring
kpE in a natural water and kDE in pure water,
kIE can be calculated.
(4) Two protocols have been written that measure kDE in
sunlight or predict kDE in sunlight from laboratory
measurements with monochromatic light (USEPA (1984) under paragraph
(f)(14) and (15) of this section; Mill et al. (1981) under paragraph
(f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of
this section; Mill et al. (1983) under paragraphs (f)(11) of this
section). As a preface to the use of the present protocol, it is not
necessary to know kDE; it will be determined under conditions
that definitively establish whether kIE is significant with
respect to kDE.
(5) This protocol provides a cost effective test method for
measuring kIE for test chemicals in a natural water
(synthetic humic water, SHW) derived from commercial humic material. It
describes the preparation and standardization of SHW. To implement the
method, a test chemical is exposed to sunlight in round tubes containing
SHW and tubes containing pure water for defined periods of time based on
a screening test.
(6) To correct for variations in solar irradiance during the
reaction period, an actinometer is simultaneously insolated. From these
data, an indirect photoreaction rate constant is calculated that is
applicable to clear-sky, near-surface, conditions in fresh water bodies.
[[Page 47]]
(7) In contrast to kDE, which, once measured, can be
calculated for different seasons and latitudes, kIE only
applies to the season and latitude for which it is determined. This
condition exists because the solar action spectrum for indirect
photoreaction in humic-containing waters is not generally known and
would be expected to change for different test chemicals. For this
reason, kpE, which contains kIE, is likewise valid
only for the experimental data and latitude.
(8) The value of kpE represents an atypical quantity
because kIE will change somewhat from water body to water
body as the amount and quality of dissolved aquatic humic substances
change. Studies have shown, however, that for optically-matched natural
waters, these differences are usually within a factor of two (Zepp et
al. (1981) under paragraph (f)(17) of this section).
(9) This protocol consists of three separate phases that should be
completed in the following order: In Phase 1, SHW is prepared and
adjusted; in Phase 2, the test chemical is irradiated in SHW and pure
water (PW) to obtain approximate sunlight photoreaction rate constants
and to determine whether direct and indirect photoprocesses are
important; in Phase 3, the test chemical is again irradiated in PW and
SHW. To correct for photobleaching of SHW and also solar irradiance
variations, tubes containing SHW and actinometer solutions are exposed
simultaneously. From these data kpE is calculated that is the
sum of kIE and kDE (Equation 1) (Winterle and Mill
(1985) under paragraph (f)(12) of this section).
(b) Phase 1--Preparation and standardization of synthetic natural
water--(1) Approach. (i) Recent studies have demonstrated that natural
waters can promote the indirect (or sensitized) photoreaction of
dissolved organic chemicals. This reactivity is imparted by dissolved
organic material (DOM) in the form of humic substances. These materials
absorb sunlight and produce reactive intermediates that include singlet
oxygen (\1\02) (Zepp et al. (1977) under paragraph (f)(20) of
this section, Zepp et al. (1981) under paragraph (f)(17) of this
section, Zepp et al. (1981) under paragraph (f)(18) of this section,
Wolff et al. (1981) under paragraph (f)(16) of this section, Haag et al.
(1984) under paragraph (f)(6) of this section, Haag et al. (1984) under
paragraph (f)(7) of this section); peroxy radicals (RO2-)
(Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al.
(1983) under paragraph (f)(8) of this section); hydroxyl radicals (HO-)
(Mill et al. (1981) under paragraph (f)(9) of this section, Draper and
Crosby (1981, 1984) under paragraphs (f)(3) and (4) of this section);
superoxide anion (02--) and hydroperoxy radicals
(HO-). (Cooper and Zika (1983) under paragraph (f)(1) of this section,
Draper and Crosby (1983) under paragraph (f)(2) of this section); and
triplet excited states of the humic substances (Zepp et al. (1981) under
paragraph (f)(17) of this section, Zepp et al. (1985) under paragraph
(f)(21) of this section). Synthetic humic waters, prepared by extracting
commercial humic or fulvic materials with water, photoreact similarly to
natural waters when optically matched (Zepp et al. (1981) under
paragraphs (f)(17) and (18) of this section).
(ii) The indirect photoreactivity of a chemical in a natural water
will depend on its response to these reactive intermediates, and
possibly others yet unknown, as well as the ability of the water to
generate such species. This latter feature will vary from water-to-water
in an unpredictable way, judged by the complexity of the situation.
(iii) The approach to standardizing a test for indirect
photoreactivity is to use a synthetic humic water (SHW) prepared by
water-extracting commercial humic material. This material is
inexpensive, and available to any laboratory, in contrast to a specific
natural water. The SHW can be diluted to a dissolved organic carbon
(DOC) content and uv-visible absorbance typical of most surface fresh
waters.
(iv) In recent studies it has been found that the reactivity of SHW
mixtures depends on pH, and also the history of sunlight exposure (Mill
et al. (1983) under paragraph (f)(11) of this section). The SHW
solutions initially photobleach with a time-dependent rate constant. As
such, an SHW test system has been designed that is buffered to maintain
pH and is pre-aged in sunlight to produce, subsequently, a predictable
bleaching behavior.
[[Page 48]]
(v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to
a standard mixture under defined, reproducible conditions.
(2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a
clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters
of 0.1 percent NaOH solution. A stir bar is added to the flask, the
flask is capped, and the solution is stirred for 1 hour at room
temperature. At the end of this time the dark brown supernatant is
decanted off and either filtered through coarse filter paper or
centrifuged and then filtered through 0.4 )m microfilter. The pH is
adjusted to 7.0 with dilute H2SO4 and filter
sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter
Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the
absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm
and 0.7 at 370 nm.
(ii) Pre-aging is accomplished by exposing the concentrated solution
in the 2-liter flask to direct sunlight for 4 days in early spring or
late fall; 3 days in late spring, summer, or early fall. At this time
the absorbance of the solution is measured at 370 nm, and a dilution
factor is calculated to decrease the absorbance to 0.50 in a 1 cm path
length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the
mixture is brought to exact dilution with a precalculated volume of
reagent-grade water to give a final absorbance of 0.500 in a 1-cm path
length cell at 370 nm. It is tightly capped and refrigerated.
(iii) This mixture is SHW stock solution. Before use it is diluted
10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with
an absorbance of 5.00x10-2 at 370 nm, and a dissolved organic
carbon of about 5 ppm. Such values are characteristic of many surface
fresh waters.
(3) Rationale. The foregoing procedure is designed to produce a
standard humic-containing solution that is pH controlled, and
sufficiently aged that its photobleaching first-order rate constant is
not time dependent. It has been demonstrated that after 7 days of winter
sunlight exposure, SHW solutions photobleached with a nearly constant
rate constant (Mill et al. (1983) under paragraph (f)(11) of this
section).
(c) Phase 2--Screening test--(1) Introduction and purpose. (i) Phase
2 measurements provide approximate solar photolysis rate constants and
half-lives of test chemicals in PW and SHW. If the photoreaction rate in
SHW is significantly larger than in PW (factor of 2X) then
the test chemical is subject to indirect photoreaction and Phase 3 is
necessary. Phase 2 data are needed for more accurate Phase 3
measurements, which require parallel solar irradiation of actinometer
and test chemical solutions. The actinometer composition is adjusted
according to the results of Phase 2 for each chemical, to equalize as
much as possible photoreaction rate constants of chemical in SHW and
actinometer.
(ii) In Phase 2, sunlight photoreaction rate constants are measured
in round tubes containing SHW and then mathematically corrected to a
flat water surface geometry. These rate constants are not corrected to
clear-sky conditions.
(2) Procedure. (i) Solutions of test chemicals should be prepared
using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and
reagent-grade (or purer) chemicals.\1\ Reaction mixtures should be
prepared with chemicals at concentrations at less than one-half their
solubility in pure water and at concentrations such that, at any
wavelengths above 290 nm, the absorbance in a standard quartz sample
cell with a 1-cm path length is less than 0.05. If the chemicals are too
insoluble in water to permit reasonable handling or analytical
procedures, 1-volume percent acetonitrile may be added to the buffer as
a cosolvent.
---------------------------------------------------------------------------
\1\ The water should be ASTM Type IIA, or an equivalent grade.
---------------------------------------------------------------------------
(ii) This solution should be mixed 9.00:1.00 by volume with PW or
SHW stock solution to provide working solutions. In the case of SHW, it
gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each
working solution should then be transferred to separate 12 x 100 mm
quartz tubes with screw tops and tightly sealed with Mininert valves.\2\
Twenty four tubes are required for each chemical solution
[[Page 49]]
(12 samples and 12 dark controls), to give a total of 48 tubes.
---------------------------------------------------------------------------
\2\ Mininert Teflon sampling vials are available from Alltech
Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004.
---------------------------------------------------------------------------
(iii) The sample tubes are mounted in a photolysis rack with the
tops facing geographically north and inclined 30[deg] from the
horizontal. The rack should be placed outdoors over a black background
in a location free of shadows and excessive reflection.
(iv) Reaction progress should be measured with an analytical
technique that provides a precision of at least 5
percent. High pressure liquid chromatography (HPLC) or gas chromatograph
(GC) have proven to be the most general and precise analytical
techniques.
(v) Sample and control solution concentrations are calculated by
averaging analytical measurements for each solution. Control solutions
should be analyzed at least twice at zero time and at other times to
determine whether any loss of chemical in controls or samples has
occurred by some adventitious process during the experiment.
(vi) Whenever possible the following procedures should be completed
in clear, warm, weather so that solutions will photolyze more quickly
and not freeze.
(A) Starting at noon on day zero, expose to sunlight 24 sample tubes
mounted on the rack described above. Tape 24 foil-wrapped controls to
the bottom of the rack.
(B) Analyze two sample tubes and two unexposed controls in PW and
SHW for chemical at 24 hours. Calculate the round tube photolysis rate
constants (kp)SHW and (kp)W
if the percent conversions are J 20 percent but F 80 percent. The rate
constants (kp)SHW and (kp)W
are calculated, respectively, from Equations 2 and 3:
Equation 2
(kp)SHW=(1/t)Pn(Co/
Ct)SHW (in d-1)
Equation 3
(kp)W=(1/t)Pn(Co/
Ct)W (in d-1),
where the subscript identifies a reaction in SHW or PW; t is the
photolysis time in calendar days; Co is the initial molar
concentration; and Ct is the molar concentration in the
irradiated tube at t. In this case t=1 day.
(C) If less than 20 percent conversion occurs in SHW in 1 day,
repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16
days, or until 20 percent conversion is reached. Do not extend the
experiment past 16 days. If less than 20 percent photoreaction occurs in
SHW at the end of 16 days the chemical is ``photoinert''. Phase 3 is not
applicable.
(D) If more than 80 percent photoreaction occurs at the end of day 1
in SHW, repeat the experiment with eight each of the remaining foil-
wrapped PW and SHW controls. Divide these sets into four sample tubes
each, leaving four foil-wrapped controls taped to the bottom of the
rack.
(1) Expose tubes of chemical in SHW and PW to sunlight starting at
0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours.
Analyze all tubes the next day.
(2) Extimate (kp)SHW for the first tube in
which photoreaction is J 20 percent but F 80 percent. If more than 80
percent conversion occurs in the first SHW tube, report: ``The half-life
is less than one hour'' and end all testing. The chemical is
``photolabile.'' Phase 3 is not applicable.
(3) The rate constants (kp)SHW and
(kp)W are calculated from equations 2 and 3 but
the time of irradiation must be adjusted to reflect the fact that day-
averaged rate constants are approximately one-third of rate constants
averaged over only 8 daylight hours. For 1 hour of insolation enter
t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours
enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing.
(4) Once (kp)SHW and
(kp)W are measured, determine the ratio R from
equation 4:
Equation 4
R=(kp)SHW/(kp)W.
The coefficient R, defined by Equation 4, is equal to
[(kI+kD)/kD]. If R is in the range 0 to
1, the photoreaction is inhibited by the synthetic humic water and Phase
3 does not apply. If R is in the range 1 to 2, the test chemical is
marginally susceptable to indirect photolysis. In this case, Phase 3
studies are optional. If R is greater than 2,
[[Page 50]]
Phase 3 measurements are necessary to measure kpE and to
evaluate kIE.
(vii) Since the rate of photolysis in tubes is faster than the rate
in natural water bodies, values of near-surface photolysis rate
constants in natural and pure water bodies, kpE and
kDE, respectively, can be obtained from
(kp)SHW and (kp)W from
Equations 5 and 6:
Equation 5
kpE=0.45(kp)SHW
Equation 6
kDE=0.45(kp)W.
The factor 0.45 is an approximate geometric correction for scattered
light in tubes versus horizontal surfaces. A rough value of
kIE, the rate constant for indirect photolysis in natural
waters or SHW, can be estimated from the difference between
kpE and kDE using Equation 7:
Equation 7
kIE=kpE-kDE.
(3) Criteria for Phase 2. (i) If no loss of chemical is found in
dark control solutions compared with the analysis in tubes at zero time
(within experimental error), any loss of chemical in sunlight is assumed
to be due to photolysis, and the procedure provides a valid estimate of
kpE and kDE. Any loss of chemical in the dark-
control solutions may indicate the intervention of some other loss
process such as hydrolysis, microbial degradation, or volatilization. In
this case, more detailed experiments are needed to trace the problem and
if possible eliminate or minimize the source of loss.
(ii) Rate constants determined by the Phase 2 protocol depend upon
latitude, season, and weather conditions. Note that
(kp)SHW and kD values apply to round
tubes and kpE and kDE values apply to a natural
water body. Because both (kp)SHW and kD
are measured under the same conditions the ratio
((kp)SHW/kD) is a valid measure of the
susceptibility of a chemical to indirect photolysis. However, since SHW
is subject to photobleaching, (kp)SHW will
decrease with time because the indirect rate will diminish. Therefore, R
2 is considered to be a conservative limit because
(kp)SHW will become systematically smaller with
time.
(4) Rationale. The Phase 2 protocol is a simple procedure for
evaluating direct and indirect sunlight photolysis rate constants of a
chemical at a specific time of year and latitude. It provides a rough
rate constant for the chemical in SHW that is necessary for Phase 3
testing. By comparison with the direct photoreaction rate constant, it
can be seen whether the chemical is subject to indirect photoreaction
and whether Phase 3 tests are necessary.
(5) Scope and limitations. (i) Phase 2 testing separates test
chemicals into three convenient categories: ``Photolabile'',
``photoinert'', and those chemicals having sunlight half-lives in round
tubes in the range of 1 hour to 50 days. Chemicals in the first two
categories fall outside the practical limits of the test, and cannot be
used in Phase 3. All other chemicals are suitable for Phase 3 testing.
(ii) The test procedure is simple and inexpensive, but does require
that the chemical dissolve in water at sufficient concentrations to be
measured by some analytical technique but not have appreciable
absorbance in the range 290 to 825 nm. Phase 2 tests should be done
during a clear-sky period to obtain the best results. Testing will be
less accurate for chemicals with half-lives of less than 1 day because
dramatic fluctuations in sunlight intensity can arise from transient
weather conditions and the difficulty of assigning equivalent reaction
times. Normal diurnal variations also affect the photolysis rate
constant. Phase 3 tests should be started as soon as possible after the
Phase 2 tests to ensure that the (kp)SHW estimate
remains valid.
(6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH
7.0 buffer. The solution was filtered through a 0.2 )m filter, air
saturated, and analyzed. It contained 1.7x10 -5 M A, five-
fold less than its water solubility of 8.5x10 -5 M at 25
[deg]C. A uv spectrum (1-cm path length) versus buffer blank showed no
absorbance greater than 0.05 in the wavelength interval 290 to 825 nm, a
condition required for the Phase 2 protocol. The 180 mL mixture was
diluted
[[Page 51]]
by the addition of 20 mL of SHW stock solution.
(ii) The SHW solution of A was photolyzed in sealed quartz tubes
(12x100 mm) in the fall season starting on October 1. At the end of 1
and 2 days, respectively, the concentration of A was found to be 1.13x10
-5 M and 0.92x10 -5 M compared to unchanged dark
controls (1.53x10 -5 M).
(iii) The tube photolysis rate constant of chemical A was calculated
from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first
time point at day 1 was used because the fraction of A remaining was in
the range 20 to 80 percent:
(kp)SHW=(1/1d)Pn(1.53x10 -5/1.13 x10
-5) (kp)SHW=0.30 d-1.
(iv) From this value, kpE was found to be 0.14 d-\1\
using equation 5 under paragraph (c)(2)(vii) of this section:
kpE=0.45(0.30 d-1)=0.14d-1.
(v) From measurements in pure water, kD for chemical A
was found to be 0.085 d-1. Because the ratio of
(kp)SHW/kD(=3.5) is greater than 2,
Phase 3 experiments were started.
(d) Phase 3--Indirect photoreaction with actinometer: Calculation of
kIE and kpE--(1) Introduction and purpose.
(i) The purpose of Phase 3 is to measure kIo, the
indirect photolysis rate constant in tubes, and then to calculate
kpE for the test chemical in a natural water. If the
approximate (kp)SHW determined in Phase 2 is not
significantly greater than kD measured for the experiment
date of Phase 2, then Phase 3 is unnecessary because the test chemical
is not subject to indirect photoreaction.
(ii) In the case (kp)SHW is significantly
larger than kD, Phase 3 is necessary. The rate constant
(kp)SHW is used to choose an actinometer
composition that matches the actinometer rate to the test chemical rate.
Test chemical solutions in SHW and in pure water buffer are then
irradiated in sunlight in parallel with actinometer solutions, all in
tubes.
(iii) The actinometer used is the p-nitroacetophenone-pyridine
(PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph
(f)(5) of this section and is used in two EPA test guidelines (USEPA
(1984) under paragraphs (f) (14) and (15) of this section). By varying
the pyridine concentration, the PNAP photolysis half-life can be
adjusted over a range of several hours to several weeks. The starting
PNAP concentration is held constant.
(iv) SHW is subject to photobleaching that decreases its ability to
promote indirect photolysis based on its ability to absorb sunlight.
This effect will be significant when the test period exceeds a few days.
To correct for photobleaching, tubes containing SHW are irradiated in
action to the other tubes above.
(v) At any time, the loss of test chemical is given by Equation 8
assuming actinometric correction to constant light flux:
Equation 8
-(d[C]/dt)=kI[C]+kD[C].
(vi) The indirect photolysis rate constant, kI, is
actually time dependent because SHW photobleaches; the rate constant
kI, after pre-aging, obeys the formula:
Equation 9
kI=kIo exp(-kt),
in which kIo is the initial indirect photoreaction rate
constant and k is the SHW photobleaching rate constant. After
substituting equation 9 for kI in Equation 8 under paragraph
(d)(1)(v) of this section, and rearranging, one obtains
-(d[C]/[C]=kIo[exp(-kt)]dt+kD dt.
This expression is integrated to give Equation 10:
Equation 10
Pn(Co/C)SHW=(kIo/k)[1-exp(-
kt)]+kD t.
The term (kIo/k) can now be evaluated. Since in pure water,
Pn(Co/C)W=kD t, then subtracting this
equation from Equation 10 gives
Equation 11
Pn(Co/C)SHW-Pn(co/
C)W=(kIo/k)[1-exp(-kt)].
The photobleaching fraction, [1-exp(-kt)], is equivalent to the
expression [1-
[[Page 52]]
(A370/A[deg]370)], where A[deg]370 and
A370 are the absorbances at 370 nm, and are proportional to
humic sensitizer content at times zero and t. Therefore,
(kIo/k) is derived from the slope of a linear regression
using [Pn(Co/C)SHW-Pn(Co/
C)W] as the dependent variable and [1-(A370/
A[deg]370)SHW] as the independent variable.
(vii) To evaluate kIo, the parameter k has to be
evaluated under standard sunlight conditions. Therefore, the photolysis
rate constant for the PNAP/PYR actinometer (kA) is used to
evaluate k by linear regression on Equation 12:
Equation 12
Pn(A[deg]370/A370)=(k/
kA)Pn(Co/C)PNAP,
where the slope is (k/kA) and the value of kA is
calculated from the concentration of pyridine and the absorption of
light by PNAP: kA=2.2(0.0169)[PYR]ka. Values of
ka are listed in the following Table 1.
Table 1--Day Averaged Rate Constant (ka) \1\ for Sunlight Absorption by
PNAP as a Function of Season and Decadic Latitude \2\
------------------------------------------------------------------------
Season
Latitude ------------------------------
Spring Summer Fall Winter
------------------------------------------------------------------------
20[deg]N................................. 515 551 409 327
30[deg]N................................. 483 551 333 232
40[deg]N................................. 431 532 245 139
50[deg]N................................. 362 496 154 64
------------------------------------------------------------------------
\1\ ka=@ ega Lg in the units of day \-1\, (Mill et al. (1982) under
paragraph (f)(10) of this section).
\2\ For use in Equation 15 under paragraph (d)(2)(i) of this section.
The value of kIo is then given by Equation 13:
Equation 13
kIo=(kIo/k)(k/kA)kA.
(viii) To obtain kD, determine the ratio (kD/
kA) from a linear regression of Pn(Co/
C)W versus Pn(Co/C)PNAP according to
Equation 13a:
Equation 13a
Pn(Co/C)W=(kD/
kA)Pn(Co/C)PNAP.
The slope is (kD/kA), and kD is
obtained by multiplication of this slope with the known value of
kA: i.e., kD=(kD/
kA)kA.
(ix) Then, (kp)SHW values in SHW are
determined by summing kD and KIo as follows:
Equation 14
(kp)SHW=kIo+kD.
(x) Finally, kpE is calculated from the precise
relationship, Equation 5a:
Equation 5a
kpE=0.455(kp)SHW.
(2) Procedure. (i) Using the test chemical photoreaction rate
constant in round tubes, (kp) SHW' determined in
Phase 2 under paragraph (c) of this section, and the absorption rate
constant, k[alpha] found in Table 1, under paragraph (d)(1)(vii) of this
section, calculate the molar pyridine concentration required by the
PNAP/PYR actinometer using Equation 15:
Equation 15
[PYR]/M=26.9[(kp) SHW/ka].
This pyridine concentration makes the actinometer rate constant match
the test chemical rate constant.
(A) The variable ka (= @ e ga Lg)
is equal to the day-averaged rate constant for sunlight absorption by
PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al.
(1982) under paragraph (f)(10) of this section, Zepp and Cline (1977)
under paragraph (f)(19) of this section) which changes with season and
latitude.
(B) The variable ka is selected from Table 1 under
paragraph (d)(1)(vii) of this section for the season nearest the mid-
experiment date of Phase 2 studies and the decadic latitude nearest the
experimental site.
(ii) Once [PYR] is determined, an actinometer solution is prepared
by adding 1.00 mL of 1.0x10-2 M (0.165 gms/100 mL) PNAP stock
solution (in CH3 CN solvent) and the required volume, V, of
PYR to a 1 liter volumetric flask. The flask is then filled with
distilled water to give 1 liter of solution. The volume V can be
calculated from Equation 16:
Equation 16
V/mL=[PYR]/0.0124.
[[Page 53]]
The PNAP/PYR solutions should be wrapped with aluminum foil and kept out
of bright light after preparation.
(iii) The following solutions should be prepared and individually
added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes
should be filled with each solution:
(A) PNAP/PYR actinometer solution.
(B) Test chemical in pH 7.0, 0.010 M phosphate buffer.
(C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW.
(D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are
wrapped in foil and used as controls.
(iv) The tubes are placed in the photolysis rack (Phase 2,
Procedure) at 0900 hours on day zero, with the controls taped to the
bottom of the rack. One tube of each composition is removed, along with
their respective controls, according to a schedule found in Table 2,
which categorizes sampling times on the basis of
(kp)SHW determined in Phase 1.
Table 2--Category and Sampling Procedure for Test and Actinometry
Solutions
------------------------------------------------------------------------
Category kp (d-1)SHW Sampling procedure
------------------------------------------------------------------------
A.............................. 5.5 J Kp J 0.69 Sample at 0, 1, 2,
4, and 8h.
B.............................. 0.69 kp Sample at 0, 1, 2,
J 0.017 4, and 8d.
C.............................. 0.17 kp Sample at 0, 4, 8,
J 0.043 16, and 32d.
------------------------------------------------------------------------
(v) The tubes containing PNAP, test chemical, and their controls are
analyzed for residual concentrations soon after the end of the
experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm
C18 reverse phase column and a uv detector set at 280 nm. The
mobile phase is 2 percent acetic acid, 50 percent acetonitrile and 48
percent water (2 mL/min flow rate). Tubes containing only SHW (solution
D) should be analyzed by absorption spectroscopy at 370 nm after storage
at 4 [deg]C in the dark. The absorbance range to be measured is 0.05 to
0.01 AU (1 cm).
(vi) If controls are well-behaved and show no significant loss of
chemical or absorbance change, then kI can be calculated. In
tabular form (see Table 4 under paragraph (d)(6)(iii)(A) of this
section) arrange the quantities Pn(Co/Ct)
SHW, Pn(Co/Ct)SHW, [1-
(A370/A\o\370)], Pn(A\o\370/
A370), and Pn(Co/C)PNAP in order of
increasing time. According to Equation 11 under paragraph (d)(1)(vi) of
this section in the form of Equation 17,
Equation 17
Pn(Co/C)SHW-Pn(Co/
C)W=(kIo/k)[1-(A370/
A\o\370)],
plot the quantities [Pn(Co/Ct)SHW-
Pn(Co/Ct)W] versus the independent
variable [1-(A370/A\o\370)]. Obtain the slope (S1)
by least square linear regression. Under the assumptions of the
protocol, S1=(kIo/k).
(vii) According to Equation 12 under paragraph (d)(1)(vii) of this
section, plot the quantities Pn(A\o\370/A370)
versus the independent variable Pn(Co/
Ct)PNAP. Obtain the slope (S2) by least squares
linear regression on Equation 12 under paragraph (d)(1)(vii) of this
section. Under the assumptions of the protocol, S2=(k/kA).
(viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this
section, determine the slope (S3) by least squares linear regression.
Under the assumptions of the protocol, S3 is equal to (kD/
kA).
(ix) From Equation 18
Equation 18
kA=0.0372[PYR]ka,
calculate kA using ka values found in Table 1
under paragraph (d)(1)(vii) of this section. The value of ka
chosen must correspond to the date closest to the mid-experiment date
and latitude closest to that of the experimental site.
(x) The indirect photoreaction rate constant, kIo, is
determined using Equation 19,
Equation 19
kIo=(S1)(kA)(S2),
by incorporating the quantities kA, S1, and S2 determined as
described in paragraphs (d)(2) (ix), (vi), and (vii) of this section,
respectively.
(xi) The rate constant kD is calculated from Equation 20,
Equation 20
kD=(S3)(kA),
[[Page 54]]
using the quantities S3 and kA determined as described above.
(xii) Then, (kp)SHW is obtained by summing
kD and kIo, as described by Equation 14 in
paragraph (d)(1)(ix) of this section:
Equation 14
(kp)SHW=kIo+kD.
(xiii) Finally, kpE is obtained by multiplying
(kp) SNW by the factor 0.455, as described by
Equation 5a in paragraph (d)(1)(x) of this section:
Equation 5a
kpE=0.455 (kp)SHW
As determined, kpE is the net environmental photoreaction
rate constant. It applies to clear sky conditions and is valid for
predicting surface photoreaction rates in an average humic containing
freshwater body. It is strictly valid only for the experimental latitude
and season.
(3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed
valid if the dark controls are well behaved and show no significant loss
of chemical. In such a case, loss of test chemical in irradiated samples
is due to photoreaction.
(4) Rationale. Simultaneous irradiation of a test chemical and
actinometer provide a means of evaluating sunlight intensities during
the reaction period. Parallel irradiation of SHW solutions allows
evaluation of the extent of photobleaching and loss of sensitizing
ability of the natural water.
(5) Scope and limitations of Phase 3 protocol. Test chemicals that
are classified as having half-lives in SHW in the range of 1 hour to 50
days in Phase 2 listing are suitable for use in Phase 3 testing. Such
chemicals have photoreaction half-lives in a range accommodated by the
PNAP/PYR actinometry in sunlight and also accommodate the persistence of
SHW in sunlight.
(6) Illustrative example. (i) From Phase 2 testing, under paragraph
(c)(6)(iii) of this section, chemical A was found to have a photolysis
rate constant, (kp)SHW' of 0.30 d-1 in
fall in round tubes at latitude 33[deg] N. Using Table 1 under paragraph
(d)(1)(vii) of this section for 30[deg] N, the nearest decadic latitude,
a fall value of ka equal to 333 d-1 is found for
PNAP. Substitution of (kp)SHW and ka
into Equation 15 under paragraph (d)(2)(i) of this section gives [PYR] =
0.0242 M. This is the concentration of pyridine that gives an
actinometer rate constant of 0.30 d-1 in round tubes in fall
at this latitude.
(ii) The actinometer solution was made up by adding a volume of
pyridine (1.95 mL) calculated from equation 16 under paragraph
(d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00
mL of 1.00 x 10-2 M PNAP in acetonitrile. The flask was
filled to the mark with distilled water to give final concentrations of
[PYR]=0.0242 M and [PNAP]=1.00x10-5 M. Ten tubes of each of
the following solutions were placed in the photolysis rack at 1,200
hours on day zero:
(A) Chemical A (1.53x10-5 M) in standard SHW (0.010 M, pH
7 phosphate buffer).
(B) Chemical A (1.53x10-5), in 0.010 M, pH 7 phosphate
buffer.
(C) SHW standard solution diluted with water 0.90 to 1.00 to match
solution A.
(D) PNAP/PYR actinometer solution. Ten additional foil-wrapped
controls of each mixture were taped to the bottom of the rack.
(iii) The test chemical had been placed in category B, Table 2 under
the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2
rate constant under paragraph (c) of this section. Accordingly, two
tubes of each irradiated solution and two tubes of each blank solution
were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged
analytical results obtained at the end of the experiment are shown in
the following Table 3.
Table 3--Chemical Analytical Results for Illustrative Example, Phase 3
----------------------------------------------------------------------------------------------------------------
10\5\[C]\SHW\, 10\5\ [PNAP],
Day M 10\5\[C]\W\, M A\SHW\370 M
----------------------------------------------------------------------------------------------------------------
0............................................... 1.53 1.53 0.0500 1.00
1............................................... 1.03 1.40 0.0470 0.810
[[Page 55]]
2............................................... 0.760 1.30 0.0440 0.690
4............................................... 0.300 1.01 0.0370 0.380
8............................................... 0.130 0.800 0.0320 0.220
----------------------------------------------------------------------------------------------------------------
Data for solutions A through D are given in column 2 through 5,
respectively. No significant chemical loss was found in the dark
controls.
(A) From these items the functions Pn(Co/C)
SNW' Pn(Co/C)W' [1--(A370/
A\o\370)SNW], Pn(A\o\370/
A370), and Pn(Co/C)PNAP were
calculated, as shown in the following Table 4 which was derived from
Table 3 under paragraph (d)(6)(iii) of this section:
Table 4--Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3
----------------------------------------------------------------------------------------------------------------
1-(A 370 / Pn(A\o\370 /
Day Pn(Co/C)SHW Pn(Co/C)W A\o\370) A370) Pn(Co /C) PNAP
----------------------------------------------------------------------------------------------------------------
0............................... 0 0 0 0 0
1............................... 0.396 0.0888 0.0600 0.0618 0.211
2............................... 0.700 0.163 0.120 0.128 0.371
4............................... 1.629 0.415 0.260 0.301 0.968
8............................... 2.465 0.648 0.360 0.446 1.514
----------------------------------------------------------------------------------------------------------------
(B) Slope S1=(kIo/k) was calculated according to Equation
17 under paragraph (d)(2)(vi) of this section and was found to be 4.96
by a least squares regression with a correlation coefficient equal to
0.9980. The following Figure 1 shows a plot of Equation 17 under
paragraph (d)(2)(vi) of this section and its best-fit line.
[GRAPHIC] [TIFF OMITTED] TC01AP92.034
Figure 1--Graphic determination of S1=(kIo/k) based on
Equation 17 under paragraph (d)(2)(vi) of this section.
(C) Slope S2=(k/ka) was also derived from Table 4 under
paragraph (d)(6)(iii)(A) of this section by a fit of
Pn(A\o\370 /A370) SHW and
Pn(Co /C)PNAP to Equation 12 under paragraph
(d)(l)(vii) of this section. This plot is displayed in the following
Figure 2; the slope S2 was found to be 0.295 and the correlation
coefficient was equal to 0.9986.
[[Page 56]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.035
Figure 2--Graphic determination of S2=(k/kA) based on
Equation 12 under paragraph (d)(1)(vii) of this section.
(D) Using the data in columns 3 and 6 in Table 4 under paragraph
(d)(6)(iii)(A) of this section, slope S3 was calculated by regression
from Equation 13a under paragraph (d)(1)(viii) of this section and was
found to be 0.428 with correlation coefficient euqal to 0.99997.
(E) Using Equation 18 under paragraph (d)(2)(ix) of this section,
kA was found to be =0.300 d-1.
(F) The values of S1, S2, and kA were then combined in
Equation 19 under paragraph (d)(2)(x) of this section to give
kIo as follows:
Equation 19
kIo=(4.96)(0.300)(0.295)=0.439 d-1.
(G) The rate constant kD was calculated from the product
of S3 and kA as expressed in Equation 20 under paragraph
(d)(2)(xi) of this section as follows:
Equation 20
kD=(0.428)(0.300)=0.128d-1.
(H) The sum of kD and kIo was multiplied by
0.455 to obtain kpE as follows:
Equation 21
kpE=(0.455)(0.439+0.128)d-1=0.258 d-1.
(I) Since kpE is a first-order rate constant, the half-
life, t1/2E, is given by Equation 22:
Equation 22
t1/2E=0.693/kpE.
Substituting the value of kpE from Equation 21 under
paragraph (d)(6)(iii)(H) of this section in Equation 22 yielded
Equation 23
t1/2E=0.693/0.258d-1=2.7 d.
(e) Data and reporting--(1) Test conditions--(i) Specific analytical
and recovery procedures. (A) Provide a detailed description or reference
for the analytical procedures used, including the calibration data and
precision.
(B) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(ii) Other test conditions. (A) Report the site and latitude where
the photolysis experiments were carried out.
(B) Report the dates of photolysis, weather conditions, times of
exposure, and the duration of exposure.
(C) If acetonitrile was used to solubilize the test chemical, report
the volume percent.
(D) If a significant loss of test chemical occurred in the control
solutions for pure water and SHW, indicate the causes and how they were
eliminated or minimized.
(2) Test data report--(i) Phase 2 Screening Test under paragraph (c)
of this section. (A) Report the initial molar concentration of test
chemical, Co, in pure water and SHW for each replicate and
the mean value.
(B) Report the molar concentration of test chemical, Ct,
in pure water and SHW for each replicate and the mean value for each
time point t.
(C) Report the molar concentration of test chemical for each
replicate control sample and the mean value for each time point.
(D) Report the values of (kp)SHW and
(kp)W for the time point t in which the fraction
of test chemical photoreacted is in the range 20 to 80 percent.
(E) If small losses of test chemical were observed in SHW and pure
water, report a first-order rate constant loss,
(kp)loss. Calculate and report
(kp)obs for SHW and/or pure water. Calculate and
report the corrected first-order rate
[[Page 57]]
constant for SHW and/or pure water using the relationship expressed in
Equation 24:
Equation 24
kp=(kp)obs-
(kp)loss.
(F) Report the value of R calculated from Equation 4 under paragraph
(c)(2)(vi)(D)(4) of this section.
(G) Report the values of kpE and kDE obtained
from Equations 5 and 6, respectively under paragraph (c)(2)(vii) of this
section; report the corresponding half-life calculated from Equation 22
under paragraph (d)(6)(iii)(I) of this section.
(ii) Phase 3--Indirect photoreaction with actinometer. (A) Report
the initial molar concentration of test chemical, Co, in pure
water and in SHW for each replicate and the mean value.
(B) Report the initial absorbance A\o\370 of the SNW
solution.
(C) Report the initial molar concentration of PNAP of each replicate
and the mean value in the actinometer. Report the concentration of
pyridine used in the actinometer which was obtained from Equation 15
under paragraph (d)(2)(i) of this section.
(D) Report the time and date the photolysis experiments were
started, the time and date the experiments were completed, and the
elapsed photolysis time in days.
(E) For each time point t, report the separate values of the
absorbance of the SHW solution, and the mean values.
(F) For each time point for the controls, report the separate values
of the molar concentrations of test chemical in pure water and SHW, and
the absorbance of the SHW solution, and the mean values.
(G) Tabulate and report the following data: t, [C]\SHW\, [C]\W\,
A\SNW\370, [PNAP].
(H) From the data in (G), tabulate and report the following data: t,
Pn(Co/C)SNW, Pn(Co/C)W, [1-
(A370/A\o\370)SNW], Pn(A\o\370/
A370), Pn(Co/C)PNAP.
(I) From the linear regression analysis of the appropriate data in
step (H) in Equation 17 under paragraph (d)(2)(vi) of this section,
report the slope S1 and the correlation coefficient.
(J) From the linear regression analysis of the appropriate data in
step (H) in Equation 12 under paragraph (d)(1)(vii) of this section,
report the slope S2 and the correlation coefficient.
(K) From the linear regression analysis of the appropriate data in
step (H) in Equation 13a under paragraph (d)(1)(viii) of this section,
report the slope S3 and the correlation coefficient.
(L) If loss of chemical was observed during photolysis in pure water
and SHW, then report the data Pn(Co/C)corr,
Pn(Co/C)obs, Pn(Co/C)loss as
described in paragraph (e)(2)(E) of this section. Repeat steps (H), (I),
(J), (K) where applicable and report S1, S2, S3 and the corresponding
correlation coefficients.
(M) Report the value of the actinometer rate constant obtained from
Equation 18 under paragraph (d)(2)(ix) of this section.
(N) Report the value of kIo obtained from Equation 19
under paragraph (d)(2)(x) of this section.
(O) Report the value of kD obtained from Equation 20
under paragraph (d)(2)(xi) of this section.
(P) Report the value of (kpE)SHW, obtained
from Equation 14 under paragraph (d)(1)(ix) of this section, and the
value of kpE obtained from Equation 5a under paragraph
(d)(1)(x) of this section.
(Q) Report the half-life, t1/2E, obtained from Equation
22 under paragraph (d)(6)(iii)(I) of this section.
(f) References. For additional background information on this test
guideline the following references should be consulted.
(1) Cooper W.J., Zika R.G. ``Photochemical formation of hydrogen
peroxide in surface and ground waters exposed to sunlight.'' Science,
220:711. (1983).
(2) Draper W.M., Crosby D.G. ``The photochemical generation of
hydrogen peroxide in natural waters.'' Archives of Environmental
Contamination and Toxicology, 12:121. (1983).
(3) Draper, W.M. and Crosby D.G. ``Solar photooxidation of
pesticides in dilute hydrogen peroxide.'' Journal of Agricultural and
Food Chemistry, 32:231. (1984).
(4) Draper W.M., Crosby D.G. ``Hydrogen peroxide and hydroxyl
radical:
[[Page 58]]
Intermediates in indirect photolysis reactions in water.'' Journal of
Agricultural and Food Chemistry, 29:699. (1981).
(5) Dulin D., Mill T. ``Development and evaluation of sunlight
actinometers.'' Environmental Science and Technology, 6:815. (1982).
(6) Haag H.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part I; Furfuryl alcohol as a trapping agent.''
Chemosphere, 13:631. (1984).
(7) Haag W.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part II: Quantum yields of its production by some
natural humic materials as a function of wavelength.'' Chemosphere,
13:641. (1984).
(8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman
H., Liu A., Davenport J.E. Toxic substances process data generation and
protocol development. Work assignment 12, test standard development.
``Section 3. Indirect photolysis.'' Draft final report. EPA Contract No.
68-03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (1984).
(9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G.,
Smith J.H. ``Laboratory protocols for evaluating the fate of organic
chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4.
Oxidation in water.'' EPA 600/3-82-022. Environmental Research
Laboratory, Office of Research and Development, EPA, Athens, GA. (1981).
(10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich
V.P., Dulin D.E., Tse D.S., Lee G. ``Design and validation of screening
and detailed methods for environmental processes. Apendix C. Lower-tier
direct photolysis protocol.'' Draft final report. EPA Contract No. 68-
01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC.
(1982).
(11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H.,
Tse D., Liu A. Toxic substances process data generation and protocol
development. Work assignment 12. ``Appendix B. Upper-tier protocol for
direct photolysis in water.'' Draft final report. EPA Contract No. 68-
03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (July 1983).
(12) Winterle J.S., Mill T. Toxic substances process data generation
and protocol development. Work assignment 18. ``Indirect photoreaction
protocol.'' Draft EPA special report. EPA Contract No. 68-03-2981.
Environmental Research Laboratory, Office of Research and Development,
EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA,
Washington, DC. (1985).
(13) Mill T., Hendry D.G., Richardson H. ``Free radical oxidants in
natural waters.'' Science, 207:886. (1980).
(14) U.S. Environmental Protection Agency (USEPA), Office of
Pollution Prevention and Toxics (OPPT). ``Chemical fate test guidelines.
Test guideline (CG, CS-6000). Photolysis in aqueous solution.'' EPA-560/
6-84-003. NTIS publication PB-84-233287. (1984).
(15) USEPA, OPPT. ``Chemical fate test guidelines. Test guildeline
(CG, CS-6010). Laboratory determination of the direct photolysis
reaction quantum yield in aqueous solution and sunlight photolysis.''
EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
(16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. ``The
formation of singlet oxygen in surface waters.'' Chemosphere, 10:59.
(1981).
(17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: I. Sunlight
induced reactions of aquatic pollutants photosensitized by humic
substances.'' Chemosphere, 10:109. (1981).
(18) Zepp R.G., Baughman G.L., Schlozhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: II.
Photosensitized oxygenations.'' Chemosphere, 10:119. (1981).
(19) Zepp R.G., Cline D.M. ``Rates of direct photolysis in aquatic
environments.'' Environmental Science and Technology, 11:359. (1977).
(20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. ``Singlet
oxygen in natural waters.'' Nature, 267:421. (1977).
[[Page 59]]
(21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. ``Photosensitized
transformations involving electronic energy transfer in natural waters:
role of humic substances.'' Environmental Science and Technology, 19:74.
(1985).
[53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988]