United States Prevention, Pesticides EPA712-C-98-099
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.5270
Indirect Photolysis
Screening Test
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.5270 Indirect photolysis screening test: Sunlight pho-
tolysis in waters containing dissolved humic substances.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPPT guideline under 40 CFR
795.70 Indirect Photolysis Screening Test: Sunlight Photolysis in Waters
Containing Dissolved Humic Substances.
(b) Introduction. (1) Chemicals dissolved in natural waters are sub-
ject to two types of photoreaction. In the first case, the chemical of interest
absorbs sunlight directly and is transformed to products when unstable ex-
cited 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 di-
rect photolysis, this photoreaction is governed initially by the spectroscopic
properties of the natural water. In general, both indirect and direct proc-
esses can proceed simultaneously. Under favorable conditions the measure-
ment 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 (kon) and indirect (km) pathways which can
be expressed by the relationship
Equation 1
kpE = kDE + km
This relationship is obtained when the reaction volume is optically thin
so that a negligible fraction of the incident light is absorbed and is suffi-
ciently dilute in test chemical; thus the direct and indirect photoreaction
processes become first-order.
(2) In pure water only, direct photoreaction is possible, although hy-
drolysis, biotransformation, sorption, and volatilization also can decrease
the concentraton of a test chemical. By measuring kpn in a natural water
and kDE in pure water, km can be calculated.
(3) Two protocols have been written that measure koE in sunlight
or predict koE in sunlight from laboratory measurements with
monochromatic light (USEPA (1984) under paragraphs (g)(14) and (g)(15)
of this guideline; Mill et al. (1981) under paragraph (g)(9) of this guide-
line; Mill et al. (1982) under paragraph (g)(10) of this guideline; Mill et
al. (1983) under paragraph (g)(ll) of this guideline). As a preface to the
use of the present protocol, it is not necessary to know kos; it will be
determined under conditions that definitively establish whether km is sig-
nificant with respect to
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(4) This protocol provides a cost effective test method for measuring
km 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 ex-
posed to sunlight in round tubes containing SHW and tubes containing
pure water for defined periods of time based on a screening test.
(5) 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.
(6) In contrast to kDE, which, once measured, can be calculated for
different seasons and latitudes, km 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 gen-
erally known and would be expected to change for different test chemicals.
For this reason, kpE, which contains km, is likewise valid only for the
experimental data and latitude.
(7) The value of kpE represents an atypical quantity because km will
change somewhat from water body to water body as the amount and qual-
ity of dissolved aquatic humic substances change. Studies have shown,
however, that for optically-matched natural waters, these differences are
usually within a factor of 2 (Zepp et al. (1981) under paragraph (g)(17)
of this guideline).
(8) This protocol consists of three separate phases that should be com-
pleted 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 deter-
mine 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 contain-
ing SHW and actinometer solutions are exposed simultaneously. From
these data kpE is calculated that is the sum of km and koE (Equation 1)
(Winterle and Mill (1985) under paragraph (g)(12) of this guideline).
(c) 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 mate-
rial (DOM) in the form of humic substances. These materials absorb sun-
light and produce reactive intermediates that include singlet oxygen (^2)
(Zepp et al. (1977) under paragraph (g)(20) of this guideline, Zepp et al.
(1981) under paragraph (g)(17) of this guideline, Zepp et al. (1981) under
paragraph (g)(18) of this guideline, Wolff et al. (1981) under paragraph
(g)(16) of this guideline, Haag et al. (1984) under paragraph (g)(6) of this
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guideline, Haag et al. (1984) under paragraph (g)(7) of this guideline);
peroxy radicals (RXV) (Mill et al. (1981) under paragraph (g)(9) of this
guideline; Mill et al. (1983) under paragraph (g)(8) of this guideline);
hydroxyl radicals (HO) (Mill et al. (1981) under paragraph (g)(9) of this
guideline, Draper and Crosby (1981, 1984) under paragraphs (g)(3) and
(g)(4) of this guideline); superoxide anion (62 •) and hydroperoxy radicals
(HO-). (Cooper and Zika (1983) under paragraph (g)(l) of this guideline,
Draper and Crosby (1983) under paragraph (g)(2) of this guideline); and
triplet excited states of the humic substances (Zepp et al. (1981) under
paragraph (g)(17) of this guideline, Zepp et al. (1985) under paragraph
(g)(21) of this guideline). 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
(g)(17) and (g)(18) of this guideline).
(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 (g)(ll) of this guideline. 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.
(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 is added to
a clean 2 L Pyrex Erlenmeyer flask. The flask is filled with 2 L 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 h 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
(im microfilter. The pH is adjusted to 7.0 with dilute H2SO4 and filter
sterilized through a 0.2 (im filter into a rigorously cleaned 2-L Erlenmeyer
flask. This mixture contains roughly 60 ppm DOC and the absorbance
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(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-L 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 readjusted 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 ten-
fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with
an absorbance of 5.00 x 10 2 at 370 nm, and a (DOC) 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 (g)(ll) of this guideline).
(d) 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. (The water should be ASTM Type IIA, or
an equivalent grade). 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 reason-
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able handling or analytical procedures, 1-volume percent acetonitrile may
be added to the buffer as a cosolvent.
(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 tenfold dilution of SHW stock solution. Aliquots (6 mL) 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.
(Mininert Teflon sampling vials are available from Alltech Associates,
Inc., 202 Campus Dr., Arlington Heights, IL 60004.) Twenty-four tubes
are required for each chemical solution (12 samples and 12 dark controls),
to give a total of 48 tubes.
(iii) The sample tubes are mounted in a photolysis rack with the tops
facing geographically north and inclined 30° 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 tech-
nique that provides a precision of at least + 5 percent. High pressure liquid
chromatography (HPLC) or gas chromatography (GC) have proven to be
the most general and precise analytical techniques.
(v) Sample and control solution concentrations are calculated by aver-
aging 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 h. 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 = (l/t)Pn(Co/Ct)sHw (in (H)
Equation 3
(kp)w = (l/t)Pn(Co/Ct)w (in (H)
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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 time 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.
(7) 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 equa-
tions 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 h of insolation enter
t = 0.125 day into equation 2. For reaction times of 2, 4, and 8 h 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 susceptible to indirect photolysis. In this
case, Phase 3 studies are optional. If R > 2, Phase 3 measurements are
necessary to measure kpn and to evaluate kin.
(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, kpn and koE, respectively, can be obtained
from (kp)sHw and (kp)w from Equations 5 and 6:
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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 km, 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
km = 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 kpn and kon-
Any loss of chemical in the dark-control solutions may indicate the inter-
vention of some other loss process such as hydrolysis, microbial degrada-
tion, 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 ko values
apply to round tubes and kpn and koE values apply to a natural water
body. Because both (kp)SHw and kD are measured under the same condi-
tions the ratio ((kp)sHw/ko) 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 evaluat-
ing 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 h
to 50 days. Chemicals in the first two categories fall outside the practical
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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 meas-
ured 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 esti-
mate 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 (im filter, air satu-
rated, and analyzed. It contained 1.7 x 10 5 M A, fivefold less than its
water solubility of 8.5 x 10 5 M at 25 °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 by the addition of 20 mL
of SHW stock solution.
(ii) The SHW solution of A was photolyzed in sealed quartz tubes
(12^E100 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 1.13
x 10 5 M and 0.92 x 10 5 M compared to unchanged dark controls
(1.53^10 5M).
(iii) The tube photolysis rate constant of chemical A was calculated
from Equation 2 under paragraph (d)(2)(vi)(B) of this guideline. 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 = (l/ld)Pn(1.53 x 10 5/1.13 x 10 5) (kp)SHw = 0.30 cH
(iv) From this value, kpE was found to be 0.14 d-1 using equation
5 under paragraph (d)(2)(vii) of this guideline:
kpE = 0.45(0.30 d-1) = 0.14(H
(v) From measurements in pure water, kD for chemical A was found
to be 0.085 d-1. Because the ratio of (kp)sHw/ko(= 3.5) is greater than
2, Phase 3 experiments were started.
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(e) Phase 3—Indirect photoreaction with actinometer: Calculation
of km and kpn—(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 kpn for the test chemical in a natural water. If the approximate
(kp)sHw determined in Phase 2 is not significantly greater than kD meas-
ured for the experiment date of Phase 2, then Phase 3 is unnecessary be-
cause the test chemical is not subject to indirect photoreaction.
(ii) In the case (kp)sHw is significantly larger than ko, 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 irradi-
ated in sunlight in parallel with actinometer solutions, all in tubes.
(iii) The actinometer used is the /7-nitroacetophenone-pyridine (PNAP/
PYR) system developed by Dulin and Mill (1982) under paragraph (g)(5)
of this guideline and is used in two EPA test guidelines (USEPA (1984)
under paragraphs (g)(14) and (g)(15) of this guideline). 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 concentra-
tion 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 depend-
ent because SHW photobleaches; the rate constant ki, after pre-aging,
obeys the formula:
Equation 9
k: = 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 (e)(l)(v) of this guideline, and rearrang-
ing, one obtains
= kio[exp(-kt)]dt + kDdt
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This expression is integrated to give Equation 10:
Equation 10
Pn(C0/C)sHw = (kio/k)[l-exp(-kt)] + kDt
The term (kio/k) can now be evaluated. In pure water, Pn(C/C)w
then subtracting this equation from Equation 10 gives
kDt,
Equation 11
Pn(C0/C)sHw - Pn(c0/C)w = (klo/k)[l-exp(-kt)]
The photobleaching fraction, [l-exp(-kt)], is equivalent to the expression
[l-(A37o/A°37o)], where A037o and A370 are the absorbances at 370 nm,
and are proportional to humic sensitizer content at times zero and t. There-
fore, (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 [l-(A37o/A°37o)sHw] as
the independent variable.
(vii) To evaluate kio, the parameter k has to be evaluated under stand-
ard sunlight conditions. Therefore, the photolysis rate constant for the
PNAP/PYR actinometer (kA) is used to evaluate k by linear regression
on Equation 12:
Pn(A°37o/A37o) = (k/kA)Pn(Co/C)pNAp
Equation 12
where the slope is (k/kA) and the value of kA is calculated from the con-
centration 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 Latitude2
LcHILUUt;
20°N
30°N
40°N
50°N
Spring
515
483
431
362
Sea:
Summer
551
551
532
496
son
Fall
409
333
245
154
Winter
327
232
139
64
1 ka=@ egal_g in the units of day -1, (Mill et al. (1982) under paragraph (g)(10) of this
guideline).
2 For use in Equation 15 under paragraph (e)(2)(i) of this guideline.
The value of kio is then given by Equation 13:
Equation 13
10
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kio = (kio/k)(k/kA)kA
(viii) To obtain kD, determine the ratio (kD/kA) from a linear regres-
sion of Pn(C0/C)w versus Pn(C0/C)PNAp according to Equation 13 a:
Equation 13a
Pn(C0/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., ko = (ko/kA)kA.
(ix) Then, (kp)SHw values in SHW are determined by summing kD
and KIO as follows:
Equation 14
kio + kD
(x) Finally, kpE is calculated from the precise relationship, Equation
5a:
Equation 5 a
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 (d) of
this guideline, and the absorption rate constant, ka found in Table 1, under
paragraph (e)(l)(vii) of this guideline, calculate the molar pyridine con-
centration 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 (= @ egaLg) is equal to the day-averaged rate
constant for sunlight absorption by PNAP (USEPA (1984) under paragraph
(g)(14) of this guideline; Mill et al. (1982) under paragraph (g)(10) of
this guideline, Zepp and Cline (1977) under paragraph (g)(19) of this
guideline) which changes with season and latitude.
(B) The variable ka is selected from Table 1 under paragraph
(e)(l)(vii) of this guideline for the season nearest the midexperiment 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 1.0 x 102 M (0.165 gms/100 mL) PNAP stock
11
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solution (in CH3 CN solvent) and the required volume, V, of PYR to a
1-L volumetric flask. The flask is then filled with distilled water to give
1 L of solution. The volume V can be calculated from Equation 16:
Equation 16
V/mL = [PYR]/0.0124
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 sam-
pling times on the basis of (kp)snw determined in Phase 1.
Table 2—Category and Sampling Procedure for Test and Actinometry Solutions
Cat-
egory
A
B
C
kp (CH)SHW
5.5 J Kp J 0.69
0.69>kp J 0.017
0.1 7>k. J 0.043
Sampling procedure
Sample at 0, 1, 2, 4, and 8h.
Sample at 0, 1, 2, 4, and 8d.
Samole at 0. 4. 8. 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 Cig 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 ana-
lyzed by absorption spectroscopy at 370 nm after storage at 4 °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 (e)(6)(iii)(A) of this guideline) arrange the
12
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quantities Pn(C0/Ct) SHW, Pn(C0/Ct)SHw, [HA37o/A°37o)], Pn(Ao37o/
ASVO), and Pn(Co/C)pNAp in order of increasing time. According to Equa-
tion 11 under paragraph (e)(l)(vi) of this guideline in the form of Equation
17,
Equation 17
Pn(Co/C)sHw-Pn(Co/C)w = (kio/k)[l - (A37o/A°37o)]
plot the quantities [Pn(Co/Ct)sHw - Pn(Co/Ct)w] versus the independent
variable [1 - (Asvo/ASvo)]. Obtain the slope (SI) by least square linear
regression. Under the assumptions of the protocol, Sl=(kio/k).
(vii) According to Equation 12 under paragraph (e)(l)(vii) of this
guideline, plot the quantities Pn(A°37o/A37o) versus the independent vari-
able Pn(Co/Ct)pNAp. Obtain the slope (S2) by least squares linear regres-
sion on Equation 12 under paragraph (e)(l)(vii) of this guideline. Under
the assumptions of the protocol, S2 = (k/kA).
(viii) Then, using Equation 13a under paragraph (e)(l)(vii) of this
guideline, determine the slope (S3) by least squares linear regression.
Under the assumptions of the protocol, S3 is equal to (ko/kA).
(ix) From Equation 18
Equation 18
kA = 0.0372[PYR]ka
calculate kA using ka values found in Table 1 under paragraph (e)(l)(vii)
of this guideline. The value of ka chosen must correspond to the date clos-
est to the midexperiment date and latitude closest to that of the experi-
mental site.
(x) The indirect photoreaction rate constant, kio, is determined using
Equation 19,
Equation 19
kio = (Sl)(kA)(S2)
by incorporating the quantities kA, SI, and S2 determined as described
in paragraphs (e)(2)(ix), (e)(2)(vi), and (e)(2)(vii) of this guideline, respec-
tively.
(xi) The rate constant kD is calculated from Equation 20,
Equation 20
kD = (S3)(kA)
13
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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 (e)(l)(ix) of this guideline:
Equation 14
= kIO+kD
(xiii) Finally, kpE is obtained by multiplying (kp) SNW by the factor
0.455, as described by Equation 5a in paragraph (e)(l)(x) of this guideline:
Equation 5 a
kpE = 0.455 (kp) SHW
As determined, kpn 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 acti-
nometer provide a means of evaluating sunlight intensities during the reac-
tion 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 h 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
(d)(6)(iii) of this guideline, chemical A was found to have a photolysis
rate constant, (kp) SHW' of 0.30 d * in fall in round tubes at latitude 33°N.
Using Table 1 under paragraph (e)(l)(vii) of this guideline for 30°N, the
nearest decadic latitude, a fall value of ka equal to 333 d * is found for
PNAP. Substitution of (kp)SHw and ka into Equation 15 under paragraph
(e)(2)(i) of this guideline gives [PYR] = 0.0242 M. This is the concentra-
tion 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 (e)(2)(ii)
14
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of this guideline to a 1 L volumetric flask containing 1.00 mL of 1.00
JE 102 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] = 1.0(LE10 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 (1.53 x 105M) in standard SHW (0.010 M, pH 7
phosphate buffer).
(B) Chemical A (1.53 x 10 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 (e)(2)(iv) of this guideline, on the basis of its Phase 2 rate
constant under paragraph (d) of this guideline. Accordingly, two tubes of
each irradiated solution and two tubes of each blank solution were re-
moved 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
Day
0
1
2
4
8
105[CpHw,M
1.53
1.03
0.760
0.300
0.130
105[C]W=M
1.53
1.40
1.30
1.01
0.800
ASHW370
0.0500
0.0470
0.0440
0.0370
0.0320
105 [PNAP], M
1.00
0.810
0.690
0.380
0.220
Data for solutions A through D are given in column 2 through 5, respec-
tively. No significant chemical loss was found in the dark controls.
(A) From these items the functions Pn(C0/C) SNW' Pn(Co/C)w' [1—
(A37o/A°37o)sNw], Pn(A°37o/A37o), and Pn(Co/C)pNAp were calculated, as
shown in the following Table 4 which was derived from Table 3 under
paragraph (e)(6)(iii) of this guideline:
Table 4—Photoreaction Function for Illustrative Examples, Phase 3, Derived
From Table 3
Day
0
1
Pn(C0/C)sHw
0
0.396
Pn(C0/C)w
0
0.0888
HA 370
/A°370)
0
0.0600
Pn(A° 370
/A37o)
0
0.0618
Pn(C0 /C)
PNAP
0
0.211
15
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Table 4—Photoreaction Function for Illustrative Examples, Phase 3, Derived
From Table 3—Continued
Day
2
4
8
Pn(C0/C)sHw
0.700
1 629
2.465
Pn(C0/C)w
0.163
0415
0.648
HA 370
/A°370)
0.120
0260
0.360
Pn(A° 370
/A37o)
0.128
0301
0.446
Pn(C0 1C)
PNAP
0.371
0968
1.514
(B) Slope SI = (kio/k) was calculated according to Equation 17 under
paragraph (e)(2)(vi) of this guideline and was found to be 4.96 by a least
squares regression with a correlation coefficient equal to 0.9980. The fol-
lowing Figure 1 shows a plot of Equation 17 under paragraph (e)(2)(vi)
of this guideline and its best-fit line.
0.20 0.30
In (A370 /A370 )SHW
Figure 1—Graphic determination of SI = (kio/k)
based on Equation 17 under paragraph (e)(2)(vi) of this guideline.
(C) Slope S2 = (k/ka) was also derived from Table 4 under paragraph
(e)(6)(iii)(A) of this guideline by a fit of Pn(A°37o /A370) SHW and Pn(Co
/C)PNAP to Equation 12 under paragraph (e)(l)(vii) of this guideline. 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.
16
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In (Cn/C)D,
Figure 2—Graphic determination of S2 = (k/kA)
based on Equation 12 under paragraph (e)(l)(vii) of this guideline.
(D) Using the data in columns 3 and 6 in Table 4 under paragraph
(e)(6)(iii)(A) of this guideline, slope S3 was calculated by regression from
Equation 13a under paragraph (e)(l)(viii) of this guideline and was found
to be 0.428 with correlation coefficient euqal to 0.99997.
(E) Using Equation 18 under paragraph (e)(2)(ix) of this guideline,
kA was found to be = 0.300 d l.
(F) The values of SI, S2, and kA were then combined in Equation
19 under paragraph (e)(2)(x) of this guideline to give kio as follows:
Equation 19
kIO = (4.96)(0.300)(0.295) = 0.439 d
(G) The rate constant ko was calculated from the product of S3 and
kA as expressed in Equation 20 under paragraph (e)(2)(xi) of this guideline
as follows:
17
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Equation 20
kD = (0.428)(0.300) = 0.123d1
(H) The sum of ko and kio was multiplied by 0.455 to obtain
as follows:
Equation 2 1
kpE = (0.455)(0.439 + 0.128)(H = 0.258 d1
(I) Since kpn is a first-order rate constant, the half-life, ti/2E, is given
by Equation 22:
Equation 22
ti/2E = 0.693/kpE
Substituting the value of kpE from Equation 21 under paragraph
(e)(6)(iii)(H) of this guideline in Equation 22 yielded
Equation 23
= 0.693/0.258d 1 = 2.7d
(f) 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 preci-
sion.
(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 expo-
sure, 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 solu-
tions 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
(d) of this guideline. (A) Report the initial molar concentration of test
chemical, Co, in pure water and SHW for each replicate and the mean
value.
18
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(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)ioss. Calculate and report
(kp)obs for SHW and/or pure water. Calculate and report the corrected first-
order rate constant for SHW and/or pure water using the relationship ex-
pressed in Equation 24:
Equation 24
(F) Report the value of R calculated from Equation 4 under paragraph
(d)(2)(vi)(D)00 of this guideline.
(G) Report the values of kpE and kDE obtained from Equations 5 and
6, respectively under paragraph (d)(2)(vii) of this guideline; report the cor-
responding half-life calculated from Equation 22 under paragraph
(e)(6)(iii)(I) of this guideline.
(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 ASvo 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 pyri-
dine used in the actinometer which was obtained from Equation 15 under
paragraph (e)(2)(i) of this guideline.
(D) Report the time and date the photolysis experiments were started,
the time and date the experiments were completed, and the elapsed pho-
tolysis 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.
19
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(G) Tabulate and report the following data: t, [C]SHW, [C]w,
ASNW37o, [PNAP].
(H) From the data in (G), tabulate and report the following data: t,
Pn(C0/C)sNw, Pn(C0/C)w, [1 - (A37o/A037o)SNW], Pn(A°37o/A37o), Pn(C0/
C)pNAP-
(I) From the linear regression analysis of the appropriate data in step
(H) in Equation 17 under paragraph (e)(2)(vi) of this guideline, 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 (e)(l)(vii) of this guideline, 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 (e)(l)(viii) of this guideline, 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(C0/C)COrr, Pn(C0/C)0bs, Pn(Co/C)iOSs as
described in paragraph (e)(2)(E) of this guideline. Repeat steps (H), (I),
(J), (K) where applicable and report SI, S2, S3 and the corresponding
correlation coefficients.
(M) Report the value of the actinometer rate constant obtained from
Equation 18 under paragraph (e)(2)(ix) of this guideline.
(N) Report the value of kio obtained from Equation 19 under para-
graph (e)(2)(x) of this guideline.
(O) Report the value of ko obtained from Equation 20 under para-
graph (e)(2)(xi) of this guideline.
(P) Report the value of (kpE)sHw, obtained from Equation 14 under
paragraph (e)(l)(ix) of this guideline, and the value of kpn obtained from
Equation 5a under paragraph (e)(l)(x) of this guideline.
(Q) Report the half-life, ti/2E, obtained from Equation 22 under para-
graph (e)(6)(iii)(I) of this guideline.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Cooper W.J. and Zika R.G. Photochemical formation of hydrogen
peroxide in surface and ground waters exposed to sunlight. Science
220:711. (1983).
(2) Draper W.M. and Crosby D.G. The photochemical generation of
hydrogen peroxide in natural waters. Archives of Environmental Contami-
nation and Toxicology 12:121. (1983).
20
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(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. and Crosby D.G. Hydrogen peroxide and hydroxyl
radical: 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) Environmental Protection Agency, Office of Toxic Substances.
Chemical fate test guidelines. Test guideline (CG, CS-6000). Photolysis
in aqueous solution. EPA-560/6-84-003. NTIS publication PB-84-
233287. (1984).
(7) Environmental Protection Agency, Office of Toxic Substances.
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).
(8) Haag, H.R. et al. Singlet oxygen in surface waters—Part I;
Furfuryl alcohol as a trapping agent. Chemosphere 13:631. (1984).
(9) Haag, W.R. et al. Singlet oxygen in surface waters—Part II: Quan-
tum yields of its production by some natural humic materials as a function
of wavelength. Chemosphere 13:641. (1984).
(10) Mill, T. et al. Toxic substances process data generation and pro-
tocol development. Work assignment 12, test standard development. Sec-
tion 3. Indirect photolysis. Draft final report. EPA Contract No. 68-03-
2981. Environmental Research Laboratory, Office of Research and Devel-
opment, EPA, Athens, GA, and Office of Toxic Substances, EPA, Wash-
ington, DC. (1984).
(11) Mill, T. et al. 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 Lab-
oratory, Office of Research and Development, EPA, Athens, GA. (1981).
(12) Mill, T. et al. Design and validation of screening and detailed
methods for environmental processes. Apendix C. Lower-tier direct pho-
tolysis protocol. Draft final report. EPA Contract No. 68-01-6325. Office
of Toxic Substances, EPA, Washington, DC. (1982).
(13) Mill, T. et al. Toxic substances process data generation and pro-
tocol 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 De-
21
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velopment, EPA, Athens, GA, and Office of Toxic Substances, EPA,
Washington, DC. (July 1983).
(14) Winterle, J.S. and T. Mill. Toxic substances process data genera-
tion 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 De-
velopment, EPA, Athens, GA and Office of Toxic Substances, EPA,
Washington, DC. (1985).
(15) Mill, T. et al. Free radical oxidants in natural waters. Science
207:886. (1980).
(16) Wolff, C.J.M. et al. The formation of singlet oxygen in surface
waters. Chemosphere 10:59. (1981).
(17) Zepp, R.G. et al. Comparison of photochemical behavior of var-
ious humic substances in water: I. Sunlight induced reactions of aquatic
pollutants photosensitized by humic substances. Chemosphere 10:109.
(1981).
(18) Zepp, R.G. et al. Comparison of photochemical behavior of var-
ious humic substances in water: II. Photosensitized oxygenations. Chemo-
sphere 10:119. (1981).
(19) Zepp, R.G. and Cline D.M. Rates of direct photolysis in aquatic
environments. Environmental Science and Technology 11:359. (1977).
(20) Zepp, R.G. et al. Singlet oxygen in natural waters. Nature
267:421. (1977).
(21) Zepp, R.G. et al. Photosensitized transformations involving elec-
tronic energy transfer in natural waters: role of humic substances. Environ-
mental Science and Technology 19:74. (1985).
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