United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–98–099

January 1998

Fate, Transport and

Transformation Test

Guidelines

OPPTS 835.5270

Indirect Photolysis

Screening Test
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin 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 ‘‘Researchers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test

Guidelines.’’
1

OPPTS 835.5270 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances.

(a) Scope—(1) Applicability. This guideline is intended to meet testing 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 harmonized 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 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. 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.

(2) 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.

(3) Two protocols have been written that measure kDE in sunlight

or predict kDE 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 guideline; Mill et al. (1982) under paragraph (g)(10) of this guideline; Mill et

al. (1983) under paragraph (g)(11) of this guideline). 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.
2

(4) 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.

(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 clearsky, near-surface conditions in fresh water bodies.

(6) 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.

(7) 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 2 (Zepp et al. (1981) under paragraph (g)(17)

of this guideline).

(8) 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 (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 material (DOM) in the form of humic substances. These materials absorb sunlight and produce reactive intermediates that include singlet oxygen (1O2)

(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
3

guideline, Haag et al. (1984) under paragraph (g)(7) of this guideline);

peroxy radicals (RO2

.

) (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 (O2

–.

) and hydroperoxy radicals

(HO.

). (Cooper and Zika (1983) under paragraph (g)(1) 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)(11) 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

μm microfilter. The pH is adjusted to 7.0 with dilute H2SO4 and filter

sterilized through a 0.2 μm filter into a rigorously cleaned 2–L Erlenmeyer

flask. This mixture contains roughly 60 ppm DOC and the absorbance
4

(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 tenfold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with

an absorbance of 5.00 × 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)(11) 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 >2×) 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 clearsky conditions.

(2) Procedure. (i) Solutions of test chemicals should be prepared

using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and reagentgrade (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-
5

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 × 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 technique 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 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 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 = (1/t)Pn(C0/Ct)SHW (in d–1)

Equation 3

(kp)W = (1/t)Pn(C0/Ct)W (in d–1)
6

where the subscript identifies a reaction in SHW or PW; t is the photolysis

time in calendar days; C0 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 foilwrapped 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 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 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:
7

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 h

to 50 days. Chemicals in the first two categories fall outside the practical
8

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 clearsky 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 μm filter, air saturated, and analyzed. It contained 1.7 × 10 –5 M A, fivefold less than its

water solubility of 8.5 × 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

(12Æ100 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

× 10 –5 M and 0.92 × 10 –5 M compared to unchanged dark controls

(1.53Æ10 –5 M).

(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 = (1/1d)Pn(1.53 × 10 –5/1.13 × 10 –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 (d)(2)(vii) of this guideline:

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.
9

(e) Phase 3—Indirect photoreaction with actinometer: Calculation

of kIE and kpE—(1) Introduction and purpose. (i) The purpose of Phase

3 is to measure kI0, 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 (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 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 = kI0 exp(–kt)

in which kI0 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)(1)(v) of this guideline, and rearranging, one obtains

–(d[C]/[C] = kI0[exp(–kt)]dt + kDdt
10

This expression is integrated to give Equation 10:

Equation 10

Pn(C0/C)SHW = (kI0/k)[1–exp(–kt)] + kDt

The term (kI0/k) can now be evaluated. In pure water, Pn(C/C)W = kDt,

then subtracting this equation from Equation 10 gives

Equation 11

Pn(C0/C)SHW – Pn(c0/C)W = (kIo/k)[1–exp(–kt)]

The photobleaching fraction, [1–exp(–kt)], is equivalent to the expression

[1–(A370/A°370)], where A°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(C0/

C)SHW-Pn(C0/C)W] as the dependent variable and [1-(A370/A°370)SHW] as

the independent variable.

(vii) To evaluate kI0, 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°370/A370) = (k/kA)Pn(C0/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

Latitude

Season

Spring Summer Fall Winter

20°N ................................... 515 551 409 327

30°N ................................... 483 551 333 232

40°N ................................... 431 532 245 139

50°N ................................... 362 496 154 64

1 ka=@ egaLg in the units of day -1, (Mill et al. (1982) under paragraph (g)(10) of this

guideline).

2For use in Equation 15 under paragraph (e)(2)(i) of this guideline.

The value of kI0 is then given by Equation 13:

Equation 13
11

kI0 = (kI0/k)(k/kA)kA

(viii) To obtain kD, determine the ratio (kD/kA) from a linear regression of Pn(C0/C)W versus Pn(C0/C)PNAP according to Equation 13a:

Equation 13a

Pn(C0/C)W = (kD/kA)Pn(C0/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 KI0 as follows:

Equation 14

(kp)SHW = kI0 + 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 (d) of

this guideline, and the absorption rate constant, ka found in Table 1, under

paragraph (e)(1)(vii) of this guideline, 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 (= @ 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)(1)(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
12

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 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 J 0.017 Sample at 0, 1, 2, 4, and 8d.

C ........ 0.17>kp J 0.043 Sample 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 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 °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
13

quantities Pn(C0/Ct) SHW, Pn(C0/Ct)SHW, [1–(A370/A0

370)], Pn(A0370/

A370), and Pn(C0/C)PNAP in order of increasing time. According to Equation 11 under paragraph (e)(1)(vi) of this guideline in the form of Equation

17,

Equation 17

Pn(C0/C)SHW–Pn(C0/C)W = (kI0/k)[1 – (A370/A0

370)]

plot the quantities [Pn(C0/Ct)SHW – Pn(C0/Ct)W] versus the independent

variable [1 – (A370/A0

370)]. Obtain the slope (S1) by least square linear

regression. Under the assumptions of the protocol, S1=(kI0/k).

(vii) According to Equation 12 under paragraph (e)(1)(vii) of this

guideline, plot the quantities Pn(A0

370/A370) versus the independent variable Pn(C0/Ct)PNAP. Obtain the slope (S2) by least squares linear regression on Equation 12 under paragraph (e)(1)(vii) of this guideline. Under

the assumptions of the protocol, S2 = (k/kA).

(viii) Then, using Equation 13a under paragraph (e)(1)(vii) of this

guideline, 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 (e)(1)(vii)

of this guideline. The value of ka chosen must correspond to the date closest to the midexperiment date and latitude closest to that of the experimental site.

(x) The indirect photoreaction rate constant, kI0, is determined using

Equation 19,

Equation 19

kI0 = (S1)(kA)(S2)

by incorporating the quantities kA, S1, and S2 determined as described

in paragraphs (e)(2)(ix), (e)(2)(vi), and (e)(2)(vii) of this guideline, respectively.

(xi) The rate constant kD is calculated from Equation 20,

Equation 20

kD = (S3)(kA)
14

using the quantities S3 and kA determined as described above.

(xii) Then, (kp)SHW is obtained by summing kD and kI0, as described

by Equation 14 in paragraph (e)(1)(ix) of this guideline:

Equation 14

(kp)SHW = kI0+kD

(xiii) Finally, kpE is obtained by multiplying (kp) SNW by the factor

0.455, as described by Equation 5a in paragraph (e)(1)(x) of this guideline:

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 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–1 in fall in round tubes at latitude 33°N.

Using Table 1 under paragraph (e)(1)(vii) of this guideline for 30°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

(e)(2)(i) of this guideline 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 (e)(2)(ii)
15

of this guideline to a 1 L volumetric flask containing 1.00 mL of 1.00

Æ 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] = 1.00Æ10–5 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 × 105 M) in standard SHW (0.010 M, pH 7

phosphate buffer).

(B) Chemical A (1.53 × 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 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

Day 105[C]SHW, M 105[C]W, M ASHW370 105 [PNAP], M

0 ........................... 1.53 1.53 0.0500 1.00

1 ........................... 1.03 1.40 0.0470 0.810

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(C0/C) SNW′ Pn(C0/C)W′ [1—

(A370/A0

370)SNW], Pn(A0370/A370), and Pn(C0/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 Pn(C0/C)SHW Pn(C0/C)W 1–(A 370

/A0

370)

Pn(Ao 370

/A370)

Pn(C0 /C)

PNAP

0 ...... 0 .................... 0 ................... 0 ................. 0 .................. 0

1 ...... 0.396 ............. 0.0888 .......... 0.0600 ........ 0.0618 ......... 0.211
16

Table 4—Photoreaction Function for Illustrative Examples, Phase 3, Derived

From Table 3—Continued

Day Pn(C0/C)SHW Pn(C0/C)W 1–(A 370

/A0

370)

Pn(Ao 370

/A370)

Pn(C0 /C)

PNAP

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 = (kI0/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 following Figure 1 shows a plot of Equation 17 under paragraph (e)(2)(vi)

of this guideline and its best-fit line.

S1 = 4.96

In (Ao

370 370 /A )SHW

0 0.10 0.20 0.30 0.40

In ( C /C) In (C /C) 0 SHW 0 w _

1.0

2.0

0

0.50

Figure 1—Graphic determination of S1 = (kI0/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(A0

370 /A370) SHW and Pn(C0

/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.
17

S2 = 0.295

0 1.0 2.0

0

0.5

0.4

0.3

0.2

0.1

In (C0 /C)PNAP

In (A0370 /A370)SHW

Figure 2—Graphic determination of S2 = (k/kA)

based on Equation 12 under paragraph (e)(1)(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)(1)(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–1.

(F) The values of S1, S2, and kA were then combined in Equation

19 under paragraph (e)(2)(x) of this guideline to give kI0 as follows:

Equation 19

kI0 = (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 (e)(2)(xi) of this guideline

as follows:
18

Equation 20

kD = (0.428)(0.300) = 0.128d–1

(H) The sum of kD and kI0 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

(e)(6)(iii)(H) of this guideline in Equation 22 yielded

Equation 23

t1/2E = 0.693/0.258d–1 = 2.7 d

(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 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

(d) of this guideline. (A) Report the initial molar concentration of test

chemical, C0, in pure water and SHW for each replicate and the mean

value.
19

(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 firstorder rate 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

(d)(2)(vi)(D)(4) 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 corresponding 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, C0, in pure water and in SHW

for each replicate and the mean value.

(B) Report the initial absorbance A0

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 (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 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.
20

(G) Tabulate and report the following data: t, [C]SHW, [C]W,

ASNW370, [PNAP].

(H) From the data in (G), tabulate and report the following data: t,

Pn(C0/C)SNW, Pn(C0/C)W, [1 – (A370/A0

370)SNW]

, Pn(A0

370/A370), 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)(1)(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)(1)(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)obs, Pn(C0/C)loss as

described in paragraph (e)(2)(E) of this guideline. 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 (e)(2)(ix) of this guideline.

(N) Report the value of kI0 obtained from Equation 19 under paragraph (e)(2)(x) of this guideline.

(O) Report the value of kD obtained from Equation 20 under paragraph (e)(2)(xi) of this guideline.

(P) Report the value of (kpE)SHW, obtained from Equation 14 under

paragraph (e)(1)(ix) of this guideline, and the value of kpE obtained from

Equation 5a under paragraph (e)(1)(x) of this guideline.

(Q) Report the half-life, t1/2E, obtained from Equation 22 under paragraph (e)(6)(iii)(I) of this guideline.

(g) References. The following references should be consulted for additional 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 Contamination and Toxicology 12:121. (1983).
21

(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: Quantum 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 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 Toxic Substances, EPA, Washington, 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 Laboratory, 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 photolysis 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 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 De-
22

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 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 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 various 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 various humic substances in water: II. Photosensitized oxygenations. Chemosphere 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 electronic energy transfer in natural waters: role of humic substances. Environmental Science and Technology 19:74. (1985).