795.70—Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances.
(a) Introduction.
(1)
Chemicals dissolved in natural waters are subject to two types of photoreaction. In the first case, the chemical of interest absorbs sunlight directly and is transformed to products when unstable excited states of the molecule decompose. In the second case, reaction of dissolved chemical is the result of chemical or electronic excitation transfer from light-absorbing humic species in the natural water. In contrast to direct photolysis, this photoreaction is governed initially by the spectroscopic properties of the natural water.
(2)
In general, both indirect and direct processes can proceed simultaneously. Under favorable conditions the measurement of a photoreaction rate constant in sunlight (KpE) in a natural water body will yield a net value that is the sum of two first-order reaction rate constants for the direct (kDE) and indirect (kIE) pathways which can be expressed by the relationship
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(3)
In pure water only, direct photoreaction is possible, although hydrolysis, biotransformation, sorption, and volatilization also can decrease the concentraton of a test chemical. By measuring kpE in a natural water and kDE in pure water, kIE can be calculated.
(4)
Two protocols have been written that measure kDE in sunlight or predict kDE in sunlight from laboratory measurements with monochromatic light (USEPA (1984) under paragraph (f)(14) and (15) of this section; Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of this section; Mill et al. (1983) under paragraphs (f)(11) of this section). As a preface to the use of the present protocol, it is not necessary to know kDE; it will be determined under conditions that definitively establish whether kIE is significant with respect to kDE.
(5)
This protocol provides a cost effective test method for measuring kIE for test chemicals in a natural water (synthetic humic water, SHW) derived from commercial humic material. It describes the preparation and standardization of SHW. To implement the method, a test chemical is exposed to sunlight in round tubes containing SHW and tubes containing pure water for defined periods of time based on a screening test.
(6)
To correct for variations in solar irradiance during the reaction period, an actinometer is simultaneously insolated. From these data, an indirect photoreaction rate constant is calculated that is applicable to clear-sky, near-surface, conditions in fresh water bodies.
(7)
In contrast to kDE, which, once measured, can be calculated for different seasons and latitudes, kIE only applies to the season and latitude for which it is determined. This condition exists because the solar action spectrum for indirect photoreaction in humic-containing waters is not generally known and would be expected to change for different test chemicals. For this reason, kpE, which contains kIE, is likewise valid only for the experimental data and latitude.
(8)
The value of kpE represents an atypical quantity because kIE will change somewhat from water body to water body as the amount and quality of dissolved aquatic humic substances change. Studies have shown, however, that for optically-matched natural waters, these differences are usually within a factor of two (Zepp et al. (1981) under paragraph (f)(17) of this section).
(9)
This protocol consists of three separate phases that should be completed in the following order: In Phase 1, SHW is prepared and adjusted; in Phase 2, the test chemical is irradiated in SHW and pure water (PW) to obtain approximate sunlight photoreaction rate constants and to determine whether direct and indirect photoprocesses are important; in Phase 3, the test chemical is again irradiated in PW and SHW. To correct for photobleaching of SHW and also solar irradiance variations, tubes containing SHW and actinometer solutions are exposed simultaneously. From these data kpE is calculated that is the sum of kIE and kDE (Equation 1) (Winterle and Mill (1985) under paragraph (f)(12) of this section).
(b) Phase 1—Preparation and standardization of synthetic natural water—
(1) Approach.
Recent studies have demonstrated that natural waters can promote the indirect (or sensitized) photoreaction of dissolved organic chemicals. This reactivity is imparted by dissolved organic material (DOM) in the form of humic substances. These materials absorb sunlight and produce reactive intermediates that include singlet oxygen ( 1 02) (Zepp et al. (1977) under paragraph (f)(20) of this section, Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al. (1981) under paragraph (f)(18) of this section, Wolff et al. (1981) under paragraph (f)(16) of this section, Haag et al. (1984) under paragraph (f)(6) of this section, Haag et al. (1984) under paragraph (f)(7) of this section); peroxy radicals (RO2 −) (Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al. (1983) under paragraph (f)(8) of this section); hydroxyl radicals (HO−) (Mill et al. (1981) under paragraph (f)(9) of this section, Draper and Crosby (1981, 1984) under paragraphs (f)(3) and (4) of this section); superoxide anion (02
− −) and hydroperoxy radicals (HO−). (Cooper and Zika (1983) under paragraph (f)(1) of this section, Draper and Crosby (1983) under paragraph (f)(2) of this section); and triplet excited states of the humic substances (Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al. (1985) under paragraph (f)(21) of this section). Synthetic humic waters, prepared by extracting commercial humic or fulvic materials with water, photoreact similarly to natural waters when optically matched (Zepp et al. (1981) under paragraphs (f)(17) and (18) of this section).
(ii)
The indirect photoreactivity of a chemical in a natural water will depend on its response to these reactive intermediates, and possibly others yet unknown, as well as the ability of the water to generate such species. This latter feature will vary from water-to-water in an unpredictable way, judged by the complexity of the situation.
(iii)
The approach to standardizing a test for indirect photoreactivity is to use a synthetic humic water (SHW) prepared by water-extracting commercial humic material. This material is inexpensive, and available to any laboratory, in contrast to a specific natural water. The SHW can be diluted to a dissolved organic carbon (DOC) content and uv-visible absorbance typical of most surface fresh waters.
(iv)
In recent studies it has been found that the reactivity of SHW mixtures depends on pH, and also the history of sunlight exposure (Mill et al. (1983) under paragraph (f)(11) of this section). The SHW solutions initially photobleach with a time-dependent rate constant. As such, an SHW test system has been designed that is buffered to maintain pH and is pre-aged in sunlight to produce, subsequently, a predictable bleaching behavior.
(v)
The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to a standard mixture under defined, reproducible conditions.
(2) Procedure.
(i)
Twenty grams of Aldrich humic acid are added to a clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters of 0.1 percent NaOH solution. A stir bar is added to the flask, the flask is capped, and the solution is stirred for 1 hour at room temperature. At the end of this time the dark brown supernatant is decanted off and either filtered through coarse filter paper or centrifuged and then filtered through 0.4 )m microfilter. The pH is adjusted to 7.0 with dilute H2 SO4 and filter sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm and 0.7 at 370 nm.
(ii)
Pre-aging is accomplished by exposing the concentrated solution in the 2-liter flask to direct sunlight for 4 days in early spring or late fall; 3 days in late spring, summer, or early fall. At this time the absorbance of the solution is measured at 370 nm, and a dilution factor is calculated to decrease the absorbance to 0.50 in a 1 cm path length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the mixture is brought to exact dilution with a precalculated volume of reagent-grade water to give a final absorbance of 0.500 in a 1-cm path length cell at 370 nm. It is tightly capped and refrigerated.
(iii)
This mixture is SHW stock solution. Before use it is diluted 10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with an absorbance of 5.00×10−2 at 370 nm, and a dissolved organic carbon of about 5 ppm. Such values are characteristic of many surface fresh waters.
(3) Rationale.
The foregoing procedure is designed to produce a standard humic-containing solution that is pH controlled, and sufficiently aged that its photobleaching first-order rate constant is not time dependent. It has been demonstrated that after 7 days of winter sunlight exposure, SHW solutions photobleached with a nearly constant rate constant (Mill et al. (1983) under paragraph (f)(11) of this section).
(c) Phase 2—Screening test—
(1) Introduction and purpose.
Phase 2 measurements provide approximate solar photolysis rate constants and half-lives of test chemicals in PW and SHW. If the photoreaction rate in SHW is significantly larger than in PW (factor of > 2X) then the test chemical is subject to indirect photoreaction and Phase 3 is necessary. Phase 2 data are needed for more accurate Phase 3 measurements, which require parallel solar irradiation of actinometer and test chemical solutions. The actinometer composition is adjusted according to the results of Phase 2 for each chemical, to equalize as much as possible photoreaction rate constants of chemical in SHW and actinometer.
(ii)
In Phase 2, sunlight photoreaction rate constants are measured in round tubes containing SHW and then mathematically corrected to a flat water surface geometry. These rate constants are not corrected to clear-sky conditions.
(2) Procedure.
(i)
Solutions of test chemicals should be prepared using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and reagent-grade (or purer) chemicals. 1 Reaction mixtures should be prepared with chemicals at concentrations at less than one-half their solubility in pure water and at concentrations such that, at any wavelengths above 290 nm, the absorbance in a standard quartz sample cell with a 1-cm path length is less than 0.05. If the chemicals are too insoluble in water to permit reasonable handling or analytical procedures, 1-volume percent acetonitrile may be added to the buffer as a cosolvent.
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Footnote(s): 1 The water should be ASTM Type IIA, or an equivalent grade.
(ii)
This solution should be mixed 9.00:1.00 by volume with PW or SHW stock solution to provide working solutions. In the case of SHW, it gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each working solution should then be transferred to separate 12 × 100 mm quartz tubes with screw tops and tightly sealed with Mininert valves. 2 Twenty four tubes are required for each chemical solution (12 samples and 12 dark controls), to give a total of 48 tubes.
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Footnote(s): 2 Mininert Teflon sampling vials are available from Alltech Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004.
(iii)
The sample tubes are mounted in a photolysis rack with the tops facing geographically north and inclined 30° from the horizontal. The rack should be placed outdoors over a black background in a location free of shadows and excessive reflection.
(iv)
Reaction progress should be measured with an analytical technique that provides a precision of at least ±5 percent. High pressure liquid chromatography (HPLC) or gas chromatograph (GC) have proven to be the most general and precise analytical techniques.
(v)
Sample and control solution concentrations are calculated by averaging analytical measurements for each solution. Control solutions should be analyzed at least twice at zero time and at other times to determine whether any loss of chemical in controls or samples has occurred by some adventitious process during the experiment.
(vi)
Whenever possible the following procedures should be completed in clear, warm, weather so that solutions will photolyze more quickly and not freeze.
(A)
Starting at noon on day zero, expose to sunlight 24 sample tubes mounted on the rack described above. Tape 24 foil-wrapped controls to the bottom of the rack.
(B)
Analyze two sample tubes and two unexposed controls in PW and SHW for chemical at 24 hours. Calculate the round tube photolysis rate constants (kp )SHW and (kp )W if the percent conversions are J 20 percent but F 80 percent. The rate constants (kp )SHW and (kp )W are calculated, respectively, from Equations 2 and 3:
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(C)
If less than 20 percent conversion occurs in SHW in 1 day, repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16 days, or until 20 percent conversion is reached. Do not extend the experiment past 16 days. If less than 20 percent photoreaction occurs in SHW at the end of 16 days the chemical is “photoinert”. Phase 3 is not applicable.
(D)
If more than 80 percent photoreaction occurs at the end of day 1 in SHW, repeat the experiment with eight each of the remaining foil- wrapped PW and SHW controls. Divide these sets into four sample tubes each, leaving four foil-wrapped controls taped to the bottom of the rack.
(1) Expose tubes of chemical in SHW and PW to sunlight starting at 0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours. Analyze all tubes the next day.
(2) Extimate (kp )SHW for the first tube in which photoreaction is J 20 percent but F 80 percent. If more than 80 percent conversion occurs in the first SHW tube, report: “The half-life is less than one hour” and end all testing. The chemical is “photolabile.” Phase 3 is not applicable.
(3) The rate constants (kp )SHW and (kp )W are calculated from equations 2 and 3 but the time of irradiation must be adjusted to reflect the fact that day-averaged rate constants are approximately one-third of rate constants averaged over only 8 daylight hours. For 1 hour of insolation enter t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing.
(4) Once (kp )SHW and (kp )W are measured, determine the ratio R from equation 4:
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(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:
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(3) Criteria for Phase 2.
(i)
If no loss of chemical is found in dark control solutions compared with the analysis in tubes at zero time (within experimental error), any loss of chemical in sunlight is assumed to be due to photolysis, and the procedure provides a valid estimate of kpE and kDE. Any loss of chemical in the dark-control solutions may indicate the intervention of some other loss process such as hydrolysis, microbial degradation, or volatilization. In this case, more detailed experiments are needed to trace the problem and if possible eliminate or minimize the source of loss.
(ii)
Rate constants determined by the Phase 2 protocol depend upon latitude, season, and weather conditions. Note that (kp )SHW and kD values apply to round tubes and kpE and kDE values apply to a natural water body. Because both (kp )SHW and kD are measured under the same conditions the ratio ((kp )SHW /kD) is a valid measure of the susceptibility of a chemical to indirect photolysis. However, since SHW is subject to photobleaching, (kp )SHW will decrease with time because the indirect rate will diminish. Therefore, R >2 is considered to be a conservative limit because (kp )SHW will become systematically smaller with time.
(4) Rationale.
The Phase 2 protocol is a simple procedure for evaluating direct and indirect sunlight photolysis rate constants of a chemical at a specific time of year and latitude. It provides a rough rate constant for the chemical in SHW that is necessary for Phase 3 testing. By comparison with the direct photoreaction rate constant, it can be seen whether the chemical is subject to indirect photoreaction and whether Phase 3 tests are necessary.
(5) Scope and limitations.
(i)
Phase 2 testing separates test chemicals into three convenient categories: “Photolabile”, “photoinert”, and those chemicals having sunlight half-lives in round tubes in the range of 1 hour to 50 days. Chemicals in the first two categories fall outside the practical limits of the test, and cannot be used in Phase 3. All other chemicals are suitable for Phase 3 testing.
(ii)
The test procedure is simple and inexpensive, but does require that the chemical dissolve in water at sufficient concentrations to be measured by some analytical technique but not have appreciable absorbance in the range 290 to 825 nm. Phase 2 tests should be done during a clear-sky period to obtain the best results. Testing will be less accurate for chemicals with half-lives of less than 1 day because dramatic fluctuations in sunlight intensity can arise from transient weather conditions and the difficulty of assigning equivalent reaction times. Normal diurnal variations also affect the photolysis rate constant. Phase 3 tests should be started as soon as possible after the Phase 2 tests to ensure that the (kp )SHW estimate remains valid.
(6) Illustrative Example.
(i)
Chemical A was dissolved in 0.010 M pH 7.0 buffer. The solution was filtered through a 0.2 )m filter, air saturated, and analyzed. It contained 1.7×10 −5 M A, five-fold 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 (12×100 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 (1.53×10 −5 M).
(iii)
The tube photolysis rate constant of chemical A was calculated from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first time point at day 1 was used because the fraction of A remaining was in the range 20 to 80 percent:
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(iv)
From this value, kpE was found to be 0.14 d− 1 using equation 5 under paragraph (c)(2)(vii) of this section:
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(v)
From measurements in pure water, kD for chemical A was found to be 0.085 d−1. Because the ratio of (kp )SHW /kD (=3.5) is greater than 2, Phase 3 experiments were started.
(d)
Phase 3—Indirect photoreaction with actinometer: Calculation of kIE
and kpE —(1) Introduction and purpose.
(i)
The purpose of Phase 3 is to measure kIo, the indirect photolysis rate constant in tubes, and then to calculate kpE for the test chemical in a natural water. If the approximate (kp )SHW determined in Phase 2 is not significantly greater than kD measured for the experiment date of Phase 2, then Phase 3 is unnecessary because the test chemical is not subject to indirect photoreaction.
(ii)
In the case (kp )SHW is significantly larger than kD, Phase 3 is necessary. The rate constant (kp )SHW is used to choose an actinometer composition that matches the actinometer rate to the test chemical rate. Test chemical solutions in SHW and in pure water buffer are then irradiated in sunlight in parallel with actinometer solutions, all in tubes.
(iii)
The actinometer used is the p -nitroacetophenone-pyridine (PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph (f)(5) of this section and is used in two EPA test guidelines (USEPA (1984) under paragraphs (f) (14) and (15) of this section). By varying the pyridine concentration, the PNAP photolysis half-life can be adjusted over a range of several hours to several weeks. The starting PNAP concentration is held constant.
(iv)
SHW is subject to photobleaching that decreases its ability to promote indirect photolysis based on its ability to absorb sunlight. This effect will be significant when the test period exceeds a few days. To correct for photobleaching, tubes containing SHW are irradiated in action to the other tubes above.
(v)
At any time, the loss of test chemical is given by Equation 8 assuming actinometric correction to constant light flux:
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(vi)
The indirect photolysis rate constant, kI, is actually time dependent because SHW photobleaches; the rate constant kI, after pre-aging, obeys the formula:
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This expression is integrated to give Equation 10:
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(vii)
To evaluate kIo, the parameter k has to be evaluated under standard sunlight conditions. Therefore, the photolysis rate constant for the PNAP/PYR actinometer (kA) is used to evaluate k by linear regression on Equation 12:
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| 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=@ ega Lg in the units of day -1, (Mill et al. (1982) under paragraph (f)(10) of this section). | ||||
| 2 For use in Equation 15 under paragraph (d)(2)(i) of this section. | ||||
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(viii)
To obtain kD, determine the ratio (kD /kA) from a linear regression of Pn(Co /C)W versus Pn(Co /C)PNAP according to Equation 13a:
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(2) Procedure.
(i)
Using the test chemical photoreaction rate constant in round tubes, (kp) SHW′ determined in Phase 2 under paragraph (c) of this section, and the absorption rate constant, kα found in Table 1, under paragraph (d)(1)(vii) of this section, calculate the molar pyridine concentration required by the PNAP/PYR actinometer using Equation 15:
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This pyridine concentration makes the actinometer rate constant match the test chemical rate constant.
(A)
The variable ka (= @ e ga Lg) is equal to the day-averaged rate constant for sunlight absorption by PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al. (1982) under paragraph (f)(10) of this section, Zepp and Cline (1977) under paragraph (f)(19) of this section) which changes with season and latitude.
(B)
The variable ka is selected from Table 1 under paragraph (d)(1)(vii) of this section for the season nearest the mid-experiment date of Phase 2 studies and the decadic latitude nearest the experimental site.
(ii)
Once [PYR] is determined, an actinometer solution is prepared by adding 1.00 mL of 1.0×10−2 M (0.165 gms/100 mL) PNAP stock solution (in CH3 CN solvent) and the required volume, V, of PYR to a 1 liter volumetric flask. The flask is then filled with distilled water to give 1 liter of solution. The volume V can be calculated from Equation 16:
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:
(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.
| 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 (d)(6)(iii)(A) of this section) arrange the quantities Pn(Co /Ct) SHW, Pn(Co /Ct )SHW, [1−(A370 /A o370 )], Pn(A o370 /A370 ), and Pn(Co /C)PNAP in order of increasing time. According to Equation 11 under paragraph (d)(1)(vi) of this section in the form of Equation 17,
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(vii)
According to Equation 12 under paragraph (d)(1)(vii) of this section, plot the quantities Pn(A o370 /A370) versus the independent variable Pn(Co /Ct )PNAP. Obtain the slope (S2) by least squares linear regression on Equation 12 under paragraph (d)(1)(vii) of this section. Under the assumptions of the protocol, S2=(k/kA ).
(viii)
Then, using Equation 13a under paragraph (d)(1)(vii) of this section, determine the slope (S3) by least squares linear regression. Under the assumptions of the protocol, S3 is equal to (kD /kA ).
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(xii)
Then, (kp )SHW is obtained by summing kD and kIo, as described by Equation 14 in paragraph (d)(1)(ix) of this section:
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(xiii)
Finally, kpE is obtained by multiplying (kp) SNW by the factor 0.455, as described by Equation 5a in paragraph (d)(1)(x) of this section:
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(3) Criteria for Phase 3.
As in Phase 2, Phase 3 tests are assumed valid if the dark controls are well behaved and show no significant loss of chemical. In such a case, loss of test chemical in irradiated samples is due to photoreaction.
(4) Rationale.
Simultaneous irradiation of a test chemical and actinometer provide a means of evaluating sunlight intensities during the reaction period. Parallel irradiation of SHW solutions allows evaluation of the extent of photobleaching and loss of sensitizing ability of the natural water.
(5) Scope and limitations of Phase 3 protocol.
Test chemicals that are classified as having half-lives in SHW in the range of 1 hour to 50 days in Phase 2 listing are suitable for use in Phase 3 testing. Such chemicals have photoreaction half-lives in a range accommodated by the PNAP/PYR actinometry in sunlight and also accommodate the persistence of SHW in sunlight.
(6) Illustrative example.
(i)
From Phase 2 testing, under paragraph (c)(6)(iii) of this section, chemical A was found to have a photolysis rate constant, (kp )SHW ′ of 0.30 d−1 in fall in round tubes at latitude 33° N. Using Table 1 under paragraph (d)(1)(vii) of this section 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 (d)(2)(i) of this section gives [PYR] = 0.0242 M. This is the concentration of pyridine that gives an actinometer rate constant of 0.30 d−1 in round tubes in fall at this latitude.
(ii)
The actinometer solution was made up by adding a volume of pyridine (1.95 mL) calculated from equation 16 under paragraph (d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00 mL of 1.00 × 10−2 M PNAP in acetonitrile. The flask was filled to the mark with distilled water to give final concentrations of [PYR]=0.0242 M and [PNAP]=1.00×10−5 M. Ten tubes of each of the following solutions were placed in the photolysis rack at 1,200 hours on day zero: