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Environmental fate & pathways

Phototransformation in water

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Reference
Endpoint:
phototransformation in water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct 1995 to 9 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Study type:
direct photolysis
Qualifier:
according to
Guideline:
EPA Guideline Subdivision N 161-2 (Photodegradation Studies in Water)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C-labelled at guanidine group (position 4 of oxadiazine ring)
Analytical method:
high-performance liquid chromatography
mass spectrometry
other: Thin-Layer Chromatography (TLC), Liquid Scintillation Counting (LSC)
Details on sampling:
SAMPLING INTERVALS
Duplicate irradiated and non-irradiated samples (2 vials of each at each time point) were harvested at 0, 4, 8, 11, 14 hours and on Days 1, 3, 5, 7, 14, 21 and 30. Zero hour samples were not purged for volatile collection. The duplicate non-irradiated samples and hourly irradiated samples were purged for one hour following harvest. Day 1 through Day 30 irradiated samples were purged for one hour each day using an in-line purge system. Following volatile collection,
each sample was radioassayed including the volatile traps and the radiochemical balance calculated. In addition, surrogate samples of the irradiated and nonirradiated incubations were harvested at various time points for sterility analysis and pH measurement.

SAMPLE PURIFICATION PROCEDURE
- Partition: Samples were transferred to a separatory funnel. An equivalent volume of an appropriate solvent was added to the sample. The sample was thoroughly shaken. The solvent was transferred to a graduated cylinder. This procedure was repeated two more times. The aqueous and combined organic fractions were radioassayed.
- Concentration: Small volumes of sample were concentrated under a gentle stream of N2 using an-evaporator system. Larger volumes of sample were transferred to a silylated round bottom flask and concentrated on a Buchi RElll Rotovapor equipped with a dry ice/acetone condenser. Samples were transferred to a graduated tube. The round bottom flask was rinsed at least twice with an appropriate solvent and sonicated in a Branson 2200 table top sonicator. The rinses were added to the graduated tube. The concentrated sample was radioassayed.

SAMPLE STORAGE CONDITIONS
All samples were stored after harvest in a freezer which was maintained below -5°C. Some samples were stored temporarily in a refrigerator maintained at approximately 2°C. Most samples were analyzed immediately following harvest. Some samples were stored in the freezer up to 6 days prior to analysis.
Buffers:
Sodium acetate trihydrate (1.36 g) was added to a 1 L volumetric flask and filled to the mark with Hydro Picotech water. Approximately 100 μL of a dilute acetic acid solution was used to adjust the pH to pH 5.00. The buffer was stored in a refrigerator until needed for dosing.
Light source:
Xenon lamp
Light spectrum: wavelength in nm:
>= 200 - <= 700
Details on light source:
LIGHT MEASUREMENTS
Total intensity natural sunlight measurements were made at Novartis, Greensboro, NC at 36°5.86'N latitude and 79°56.24'W longitude using an International Light IL-1700 with a SUD400 UV/VIS detector and a Heraeus Radialux equipped with a Radialux Global Sensor. Natural and artificial spectral distributions were made using an International Light IL-1700, International Light IL-760 (power supply), and an International Light IL-790 (Kratos double monochromator). Spectral distributions were measured from 200-700 nm in 10 nm increments. The natural sunlight spectral distribution from 290-400 nm, the most photolytically relevant range, was used to set the Suntest CPS+ Units 3 and 4. A ratio of natural sunlight to artificial light of approximately 1.0 was used as an ideal state. An initial and final spectral distribution was measured for each Suntest unit.
Details on test conditions:
TEMPERATURE MEASUREMENTS
Both irradiated and non-irradiated sample temperatures were monitored by the Environmental Monitoring System (EMS) composed of an Omega thermocouple, Omega data logger and computer support. For the irradiated samples, the thermocouple was inserted into a surrogate vial containing the same amount of buffer as the sample. For the non-irradiated samples, the thermocouple was placed in the constant temperature room with the samples. In addition, the temperature and humidity were measured for the non-irradiated samples, using a Honeywell hydrothermograph.

PREPARATION OF TEST SUBSTANCE
The radiolabelled test substance was dissolved in 5 mL of acetonitrile. The test substance was sonicated for approximately 5 minutes using an Branson 2200 sonicator to insure the test substance was properly dissolved. An aliquot of the test substance solution (10 μL) was diluted with 100 μL of acetonitrile for TLC and HPLC purity assays. Just prior to dosing, four aliquots of the test substance were radioassayed in 5 mL of Ready Safe. The total number of dpm needed to dose the buffer at 10 ppm and the exact volume of test substance needed was calculated. Approximately 3.683 mL (658,896,066 dpm) of the test substance needed to dose the buffer.

PURITY OF TEST SUBSTANCE
An aliquot of the test substance solution (10 μL) was diluted with 100 μL of acetonitrile for TLC and HPLC purity analysis. An aliquot of the diluted test substance solution (28,156 dpm) was applied to each of two TLC plates and developed for two dimensional TLC). One plate was scraped and quantitated to compare to the established radiochemical purity. The second plate was used for UV cochromatography with the reference standard. Since UV light very rapidly degrades the sample, a second plate for cochromatography was necessary. An aliquot of this radiolabeled solution was also injected for HPLC analysis. Two lanes of the dose solution at pre, mid and post dose were spotted for one dimensional TLC assay. One lane was used for UV cochromatography with the reference standard. The second lane was used for quantitation to make sure the test substance had not degraded during the dosing procedure. In each case the radiochemical purity compared. Favorably with the purity established by the Chemical Synthesis Group of 97.3% on 9/21/95.

APPLICATION OF TEST SUBSTANCE
The dose solution was radioassayed to determine the exact volume of dose solution needed to equal 658,896,000 dpm (10 ppm). An aliquot of the test substance solution (3.683 mL = 658,896,066 dpm) was added to the pH 5 buffer and shaken for approximately 5 minutes. This dose solution was filtered through a 0.2 μm Nalgene® filterware unit to cold sterilize the solution. Meanwhile the Labconco Purified Clean Bench was sprayed with a 3:1 ethanol:water solution to sterilize the area. All sample vials, glassware and any utensils needed for dosing were sterilized using an AMSCO Eagle Sterilizer. The dose solution was transferred to the Labconco bench, then immediately transferred to a sterile Erlenmeyer flask to prevent any radioactivity from binding to the Nalgene® flask. The dose solution (10 mL) was pipetted into each sample vial. Each sample vial was rectangular in shape (approximately 2x2x5 cm) and made of Type 1 borosilicate glass with an open top cap and Teflon coated septum. The ppm dose rate calculated by Talisman version 1.0 software was 10.388 ppm based on 12 radioassays at pre, mid and post dose.

NON-IRRADIATED INCUBATION
The dosed sample vials were tightly capped with an open top cap and a Teflon coated septum. The cap of the sample vials were labeled with the protocol number and a unique sample number and wrapped with parafilm during the incubation phase. These non-irradiated samples were wrapped in aluminum foil and placed in a aluminum foil lined box to insure no light reached the samples. The non-irradiated samples were incubated up to 30 days in the constant temperature room (Environmental Specialties, Inc. 9-19 TC/GC Chamber). The temperature and humidity were monitored with a Honeywell hydrothermograph and the temperature was also monitored by the Environmental Monitoring System (EMS).

IRRADIATED INCUBATION
The dosed sample vials were tightly capped with an open top cap and a Teflon coated septum. The vial caps were labeled with the protocol number and a unique sample number during the photolytic process. Sterile Peek® tubing was inserted into the headspace of each vial to connect to the purge system. This purge design allowed for the irradiated samples to be purged while in the photolysis chamber. The lid of each vial was wrapped with parafilm and the vial placed on its side in a water bath to allow for maximum exposure to the artificial light source. The water bath was fed by a recirculating pump to maintain the temperature at 25 ± 1°C. The temperature of the water bath was monitored by the Environmental Monitoring System (EMS). The EMS system consisted of an Omega thermocouple, an Omega data logger and computer support. The Omega thermocouple was inserted into a surrogate vial filled with 10 mL of buffer prior to dosing. The samples were covered with a Pyrex plate and placed directly under the Suntest CPS+ Photolysis Unit (Suntest Accelerated Exposure Unit, W.C. Heraeus, Hanau, Germany). The light source was a xenon arc lamp (1.8 kW) equipped with a quartz glass dish with a selective reflecting coating and a UV glass filter. These filters and the Pyrex plate absorb wavelengths below 290 nm to simulate natural sunlight. The samples were irradiated for 12 hours per day, for up to 30 days.

UV/VISIBLE SPECTRUM
The post dose solution and weekly irradiated and nonirradiated surrogate samples were used to analyze the UV/Visible spectrum of the test substance using a Beckman DU-640 Spectrophotometer. A UV/Visible spectrum from 200 - 700 nm was generated. The "peak pick" (program to pick the two highest absorbencies and report the absorbencies at those wavelengths) and the "point pick" (program to report the absorbance at a particular wavelength) programs were used to generate the absorbance data for the λmax. The molar absorptivity was calculated using these values.

PH MEASUREMENTS
The pH was measured at pre dose and on surrogate Day 7, 14, 21 and 30 irradiated and non-irradiated samples using a Fisher Accumet 915 pH meter. A two point calibration was performed prior to measuring the sample pH with certified pH 4 and pH 7 buffered solutions.

VOLATILE COLLECTION
Zero hour samples were not purged for volatile collection. Four hour through 14 hour irradiated and all non-irradiated samples were purged immediately after harvest using the nonirradiated purge system. Day 1 through Day 30 irradiated samples were purged for one hour per day while in photolysis chamber using the irradiated (in-line) purge system. In each case, the purge system consisted of a water aspirator pump (Cole Palmer Instrument Co.), a manifold, one ethylene glycol trap, two KOH traps, a sample vial, empty scintillation vials and an air inlet. The water aspirator pump was connected to a glass manifold with multiple outlets. Outlets were fitted with Silastic® Laboratory tubing connected to flow regulators and connected to an 18 gauge needle. The volatiles were trapped by drawing air through the sample vial into a series of ethylene glycol, KOH traps and empty scintillation vials at an approximate flow rate of 19 mL/min for 1 hour. The ethylene glycol trap consisted of a 20 mL scintillation vial filled with 10 mL of ethylene glycol. The KOH traps consisted of two 20 mL scintillation vials, each filled with 10 mL of a 10% w/v aqueous KOH solution. Each volatile trap was sealed with an open top cap and a Teflon coated septum. These volatile traps were connected by Peek® 0.02" ID tubing from the head space of the first trap into the solution of the next consecutive trap. The 18 gauge needle the septum of the final For the non-irradiated samples and the 0 - 14 hour irradiated samples, a gentle negative pressure was established from the water aspirator pump, then the tube of the first trap was inserted into the head space of the sample. An open-top 25 gauge needle was immediately inserted into the headspace of the sample to provide air flow. Approximately 1 hour later the tubing and needles were quickly removed from the volatile traps and the sample. Day 1 through Day 30 irradiated samples, a gentle negative pressure was established from the water aspirator pump, then the tubing was inserted from the headspace into the trap solutions beginning from the back of the trap system. There was an air inlet with a Bactivent® filter, sterilized tubing and sterile bottle already established to make sure any incoming air was sterile. Approximately 1 hour later the tubing was raised out of the volatile trap solutions and the vacuum pump turned off.
Each volatile trap contained either 10 mL of ethylene glycol or 10 mL of 10% potassium hydroxide solution. Duplicate aliquots (500 μL) of each ethylene glycol trap and (1 mL) of each KOH trap were radioassayed in 10 mL of Ready Gel. The volatile traps were assayed only at harvest following the purge.

DETERMINATION OF RADIOCHEMICAL BALANCE
The dpm concentration was based on the average dpm of the dose solution aliquots (pre, mid and post dose). Twelve aliquots of the dose solution were assayed by LSC analysis at pre, mid and post dose periods. These assays were within ±0.4% of the mean. The total radiochemical balance for each sample is equivalent to the combined percent radiochemical balance for each fraction (volatiles and solution). Since the volatile traps were only assayed following harvest, the radiochemical balance for the volatiles represents a cumulative balance. The actual calculation for the radiochemical balance per fraction (or percent of original sample) was done in the Talisman, version 1.0, computer program.

PREPARATION AND USE OF REFERENCE STANDARDS
Subsamples of the reference standards were prepared for use by dispensing the desired quantity of standard into a tared vial and adding a measured volume of an appropriate solvent. All weight measurements were made on Mettler balances. All reference standards and their preparations were stored below -5°C in the Special Studies Unit II Freezer. Consistent TLC Rf values were used as a determination of reference standard stability. If TLC Rf values were not consistent with previous assay results, the solution was discarded and a new solution prepared. All information regarding reference standards and their preparations was recorded in a laboratory notebook.
Duration:
240 h
Temp.:
25 °C
Initial conc. measured:
2.16 mg/L
Reference substance:
no
Dark controls:
yes
Computational methods:
RATE CONSTANT AND HALF LIFE DETERMINATION
The half life and disappearance of parent are displayed graphically using two methods. The first method used Origin™ computer software, version 4.0 to graph the average percent of total dose of parent for the irradiated and non-irradiated incubations. The best fit curved line for the Origin graphs was generated. The second method used Excel computer software, version 5.0 to graph the data. The average percent of total dose of parent was entered into a worksheet and the natural log of the average percent of total dose of parent was calculated. The "Trend" function was used to generate the best fit line for the natural log of the percent of total dose of parent. Since the Origin graph indicated that parent plateaued at Day 14 for the irradiated incubations, only the data through Day 14 was used to generate the best fit line, rate constant and half life for the data in Excel. All data points were used to calculate the best fit line, rate constant and half life for the non-irradiated incubations in Excel.
Preliminary study:
Not reported
Test performance:
There was one minor temperature spike up to 26.6°C lasting approximately 20 minutes during the irradiated portion of the study. The minor temperature spike occurred at approximately Day 22 which was significantly beyond the half life of 2.29 Days. There were no significant differences between the Day 21 and Day 30 samples so the effect on the study was considered negligible.
Parameter:
max lambda
Value:
250 nm
Parameter:
max epsilon
Value:
17 000 L/(mol cm)
Remarks:
approximate value for absorptivity at 250 nm
Key result
% Degr.:
93.73
Sampling time:
30 d
Test condition:
irradiated soil
% Degr.:
0.45
Sampling time:
30 d
Test condition:
non-irradiated soil
Key result
DT50:
2.29 d
Test condition:
irradiated incubation
Remarks on result:
other: rate constant of 0.3025
DT50:
595 d
Test condition:
non-irradiated incubation
Remarks on result:
other: rate constant of 0.00116
Transformation products:
yes
Remarks:
see 'Details on results'
Details on results:
All results are provided as images in 'Attached background information'.

RECOVERY OF RADIOACTIVITY
The ppm dose rate of 10.388 was calculated based on the dpm applied at the time of dosing. The radiochemical balance (recovery after harvest) was based on the radioassay of the dose solution at the time of dosing versus the radioassay of the volatiles and the dosed sample solution following harvest. Some ethylene glycol and KOH fractions were assayed resulting in a less than MQA value (Minimum Quantifiable Amount,
VOLATILES
The percent volatiles reached a cumulative average of 1.55% of the total dose for the irradiated samples and <0.02% for the nonirradiated samples by Day 30.

RATE OF DEGRADATION OF THE PARENT MOLECULE
Component A always cochromatographed with the reference standard of the test substance by two dimensional TLC. Component A cochromatographed with the test substance reference standard by HPLC. Component A was analyzed by LC/MS in the positive ion mode. The [M+l]+ ion at m/z 292 was detected, indicating a molecular weight of 291. The daughter ion spectra of component A and the reference standard of the test substance at m/z 292 shows major fragments at m/z 211 (loss of NO2 and Cl from [M+l]+), 181 (loss of CH2O, amu =30, from the guanidine ring portion of 211), and 132 (thiazolyl ring portion of the molecule). Component A was confirmed as the test substance, since all of the fragments correspond to the reference standard of the test substance. In the irradiated incubations, Component A degraded to an average of 1.16% of total dose by Day 14 then slowly degraded to 0.45% of total dose by Day 30. In the non-irradiated incubations, Component A degraded to an average of 93.73% of total dose by Day 30.

FORMATION OF DEGRADATES
There were a total of at least 22 degradates in the irradiated samples, 5 of which were also common to the non-irradiated samples. Only two components, O and Origin (M5), exceeded 10% of total dose. Component O was proven to be multicomponent (consisting of component O1, O2 and O3). Nine components each accounted for less than 1% of total dose. M1, M7, M4, M3, component D, component K, and methylurea were all observed as minor components.
- M5: M5 is the major component in the irradiated incubations representing up to an average of 65.75% of total dose by Day 30. M5 was isolated by preparatory HPLC and subsequent fractions were combined. Isolated M5 was spotted for two dimensional TLC using Solvent System I and II. These solvent systems were used for the initial characterization and quantitation of M5. Isolated M5 was also spotted for two dimensional TLC in Solvent System III and IV since these solvent systems moved Origin into the first quadrant. M5 cochromatographed with reference standard M5 in both two dimensional TLC systems. M5 was analyzed by LC/MS in the positive ion mode. The [M+l]+ ion at m/z 116 was detected, indicating a molecular weight of 115. The daughter ion spectra of rn/z 116 shows major fragments at m/z 86 (loss of CH2O, amu = 30, from 116), 57 (loss of CH3N, arnu = 29, from 86). The MS/MS analysis confirmed the identity of M5.
- Component O: Component O was quantitated as a zone. Beginning at the 8 hour samples Component O appeared as a smear to the right of M5. By Day 30 the smear had separated some into 3 components but still not distinct enough to be quantitated as separate components. Component O represented a maximum of 14.13% of total dose by Day 14. Day 21 and 30 samples were combined and concentrated. The sample was partitioned with methylene chloride and ethyl acetate. The resulting aqueous sample was concentrated and spotted for 2-D TLC. Component O was obviously 3 minor components and was quantitated as O1, O2 and O3.

See attached tables

Validity criteria fulfilled:
not specified
Conclusions:
Degradation of the test substance under photolytic conditions in the pH 5 buffered aqueous solution followed first order kinetics. The test substance degraded with a half-life of 2.29 days and a rate constant or slope of -0.3025. The test substance did not significantly degrade under non-irradiated or hydrolytic conditions at pH 5. The predicted half-life of the test substance under these conditions was 595 days with a rate constant or slope of -0.00116. Cumulative volatiles generally ranged from 0-1.66% of total dose and therefore were considered negligible and were not further characterized. There were a total of at least 22 degradates in the irradiated samples, 5 of which were also common to the non-irradiated samples. Only two components, 0 and Origin (M5), exceeded 10% of total dose. Component O was proven to be multicomponent. Nine components each accounted for less than 1% of total dose. M1, M7, M4, M3, component D, component K, and methylurea were all observed as minor components.
Executive summary:

A GLP-compliant aqueous photolysis study was conducted with the radiolabelled test substance in accordance with U.S. EPA Environmental Fate Data Requirement 40 CFR Section 158 (Subdivision N, Series 161-2). The photodegradation rate of the test substance was studied under artificial light at approximately 10 ppm in hydrolytically stable pH 5 aqueous buffered solution. The irradiated samples were irradiated with a xenon arc lamp for 12 hours per day at an average daily intensity of approximately 410 W/m2. Samples were incubated up to 30 days as either irradiated or nonirradiated (dark control) samples. All samples were maintained at approximately 25°C. Harvest of samples were frequent enough to permit an accurate determination of the degradation half-life and to monitor the formation and decline of degradates. Degradation of the radiolabelled test substance under aqueous photolytic conditions indicated first order kinetics. the test substance degraded with a half-life of 2.29 days with a rate constant of -0.3025. The radiolabelled test substance under non-irradiated (or hydrolysis) conditions was very stable with no significant degradation during the 30 day incubation period. Parent remained at 93.73% of total dose at Day 30. The half-life was estimated for the test substance at 595 days under these conditions with a rate constant of -0.00116. Photolytic degradation of the test substance led to formation of a total of at least 22 degradates as compared to only 5 degradates in the non-irradiated samples. Volatiles accounted for up to an average of 1.55% of the total dose for the irradiated incubations and <0.02% for the non-irradiated incubations. There were 2 components, Origin and 0, in the irradiated incubations that exceeded 10% of the total applied dose. Additional chromatographic analysis indicated that Component O was multicomponent. The Origin component, which accounted for up to 65.75% of total dose by Day 30, was identified as M5. The primary degradates identified were M1, M7, M4, M3, M5, methylurea, component D and component K. Evidence from this study indicates that the test substance degrades significantly under photolytic conditions.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

The test substance degraded with a half-life of 2.29 days and a rate constant of -0.3025 (1/day) under photolytic conditions in the pH 5 buffered aqueous solution following first order kinetics.

Key value for chemical safety assessment

Half-life in water:
2.29 d

Additional information

Table: Overview of available data for the test item on phototransformation in water 

Method

Guideline / GLP

Endpoint

Value (DT50)

Comment

Reference

Irradiation

pH

Duration

Temp.

Irradiated solution (~410 W/m2)

5

30 d

25 °C

EPA Subdivision N 161-2

DT50

2.29 d

Significant degradation observed under photolytic conditions

Sparrow, 1997

Non-irradiated solution

5

30 d

25 °C

DT50

595 d