Diquat dibromide

Phototransformation in water

Link to relevant study record(s)

Reference

Endpoint:

phototransformation in water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct 2003 to 20 Nov 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Study type:

direct photolysis

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147, Agricultural Production Bureau, November 24, 2000 (revised 26th June 2001)

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals, Phototransformation of Chemicals in Water-Direct and Indirect Photolysis, Draft New Guideline, August 2000

GLP compliance:

yes

Radiolabelling:

yes

Analytical method:

high-performance liquid chromatography

mass spectrometry

other: HPLC-NMR, LSC

Details on sampling:

This study was performed using a previously sampled natural pond water (Middle Row Pond). The water had previously been sterilised using gamma irradiation by Isotron PLC. An aliquot of this water was removed from this central batch for use in this study. Duplicate vessels were analysed immediately after treatment of the third regime to act as zero time samples. For the irradiated samples, duplicate vessels were removed from the Suntest instrument after 2, 4, 12, 18, 24, 48 and 72 hours (HAT) of continuous irradiation. For the dark controls, duplicate samples were sampled at a time equivalent to the maximum irradiation period for the test. All vessels were transferred back to the laboratory and analysed immediately.

Buffers:

Not reported

Light source:

Xenon lamp

Light spectrum: wavelength in nm:

>= 300 - <= 850

Details on light source:

- Instrument: The light source used to irradiate the samples was a Heraeus Suntest Accelerated Exposure Instrument Table Unit. This instrument was is fitted with a high-powered xenon burner made from high-grade quartz. This allows the passage of radiation ranging from short-wave ultraviolet to middle-wave length infrared. A system of special mirrors and filters removes infrared radiation, but allows the passage of ultraviolet and visible light with a spectral distribution that closely approximates to D65 radiation. Hence, the emission spectrum produced is closely equivalent to the global radiation of natural sunlight. The burner is mounted in a cylindrical parabolic reflector to produce approximately parallel radiation. This unit is cooled by a double blower, which directs separate air streams over the burner and photolysis tank.

- Light intensity measurements: Light intensity measurements were taken at the start and end of the irradiation periods and the mean of these values was used in calculations. A baseless vessel with quartz lid attached and surrounded by a black cardboard collar was attached to the LI-1800 sensor optic probe when taking light intensity measurements.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test apparatus/vessels: Vessels with side arms were used for this study in order to trap volatiles. The borosilicate vessels were coated in black glass paint. Each vessel was individually covered with a quartz lid, which is suitable for general optical applications requiring good transmission in the near ultraviolet and visible range.
- Photolysis tank: Treated natural water samples in photolysis vessels were placed in a stainless
steel tank, which was designed to enable cooling water to circulate through its base. In order to maintain the temperature of the vessels at the required temperature, the cooling water was circulated using a thermostatically controlled circulator. A thin layer of glycerol was added to the surface of the tank to ensure good thermal contact between the tank and the photolysis vessels and thus minimise fluctuations in the temperature. To monitor the temperature of the water a thermocouple (Type Tl) was embedded in the photolysis vessel containing the untreated water and was positioned in the tank alongside the treated samples. A temperature meter (programmed to record at 6 hourly intervals) was used to take the temperature measurements during the photolysis period
- Details of traps for volatile: Volatile radioactivity was continuously flushed from the photolysis vessels and dark controls (air being pulled through using a peristaltic pump) and collected in liquid traps (2 x 2M NaOH).

STERILISATION
- Method: The photolysis vessels, tubing and in-line microbial filters (for maintenance of sterility during incubation) were sterilised by autoclave at 120 °C. The glass pipette, all pipette tips and storage bottles for the treatment solutions were soaked in an ethanol/water mixture (70:30 v/v). The natural pond water had previously been sterilised by Gamma irradiation. Application preparation and removal of vessels for analysis was conducted in a Microbiological Class II Safety Cabinet, which was washed with ethanol/water (70:30 v/v) prior to use.
- Checks: A sample: that had been maintained under the same conditions as the treated samples (i.e. under the xenon arc lamp at 25 °C) was sacrificed to check that sterility was maintained throughout the irradiation period for the maximum time phase carried out. The sterility of the treated natural water was checked by dispensing 0.5 mL aliquots on to plates containing a nutrient agar medium. The agar plates were then maintained at 20 ± 2 °C for a period of a minimum of 7 days prior to inspection for microbial growth. “Clean” control plates were prepared for each sterility check operation, this was prepared by exposing the agar plates to the environment of the laminar flow cabinet without the addition of sample in order to provide an indication of background levels of contamination. All sterility checks were carried out in a laminar flow cabinet using aseptic techniques.

PREPARATION OF TREATMENT SOLUTIONS
The radiolabelled cation solution was dissolved in 3 mL methanol to give the stock solution. The solution was quantified by LSC and the volume required for preparation of the treatment solution determined. The irradiation was conducted in three phases, in order to accommodate all vessels under the same lamp, hence the staggered applications and irradiation. Separate treatment solutions were prepared for each of the tests.

APPLICATION AND IIRADIATION
Each application solution was prepared in a 50 mL volumetric flask using the Gamma irradiated Middle Row Pond water. 490 µL of radiolabelled test item in methanol was added to the volumetric flask using a 250 µL Gilson pipette. (The concentration of methanol was < 1% volume) 8 mL aliquots of this application solution were transferred to each photolysis vessel using a 10 mL pipette. Aliquots (up to 5 x 100 µL) of each treatment solution were quantified by LSC (pre-application), to check the homogeneity of the treatment solutions and to determine the exact application rate. The purity of each treatment solution was determined, by HPLC, after application to confirm stability.

INCUBATION OF TREATED VESSELS
Each photolysis vessel was coated with black paint to prevent entry of reflected light into the system. The vessels were then sealed using a quartz lid and held together with clips. For the irradiated samples, duplicate vessels for the same sampling interval were connected to the same flow through arrangement. The photolysis vessels were arranged inside the temperature-controlled tank, which was placed directly under the xenon arc burner in the Suntest Accelerated Exposure Instrument. The tank was cooled using a re-circulating chiller to ensure the treated vessels were maintained at 25±2 °C. For the dark controls, the duplicate treated vessels were wrapped in aluminium foil and maintained in a constant temperature cabinet (at 25 ± 2 °C).

REPLICATION
- No. of replicates (dark): 2
- No. of replicates (irradiated): 2

PROPERTIES OF THE NATURAL WATER
- pH: 7.02
- Electrical Conductivity (µS/cm): 414
- Total Carbon (mg/L): 39.2
- Total Inorganic Carbon (mg/L): 38.9
- Total Suspended Solids (mg/L): 2.8
- Nitrate- Nitrogen (mg/L): 0.5
- Ammonium- Nitrogen (mg/L): 0.2
- Alkalinity as HCO3 (mg/L): 197.4
- Total Magnesium (mg/L): 20.8
- Total Calcium (mg/L): 47.7
- Total Iron (mg/L): < 0.1
- Total Dissolved Iron (mg/L) < 0.05
- Determination of Ferric Iron Concentration (mg/L): < 0.05
- Determination of Ferrous Iron Concentration (mg/L): < 0.05

Duration:

15 d

Temp.:

25 °C

Initial conc. measured:

0.01 mg/L

Reference substance:

no

Dark controls:

yes

Preliminary study:

Not applicable

Test performance:

See 'Details on test conditions'

Parameter:

not applicable

Key result

% Degr.:

84.2

Sampling time:

72 h

Test condition:

Sterilised natural river water (pH = 7.9), continuous irradiation with Xenon arc lamp for 3 days (39.4 W.m-2 between 300-400nm), 25°C

Key result

DT50:

2 d

Test condition:

Sterilised natural river water (pH = 7.9), continuous irradiation with Xenon arc lamp for 3 days (39.4 W.m-2 between 300 - 400 nm), 25 °C

Remarks on result:

other: Summer sunlight 50 °N

DT50:

6.5 d

Test condition:

Sterilised natural river water (pH = 7.9), continuous irradiation with Xenon arc lamp for 3 days (39.4 W.m-2 between 300 - 400nm), 25 °C

Remarks on result:

other: Tokyo spring sunlight 35 °N

Transformation products:

yes

No.:

#4

No.:

#3

No.:

#5

No.:

#2

No.:

#1

Details on results:

An overview of the results is provided in Table 1 - Table 2 in 'Any other information on results incl. tables'.

STERILITY CHECKS
No microbial or fungal growth was observed on the agar plates prepared with the test samples.

MASS BALANCE
The mass balance from the irradiated samples ranged from 88.4 - 100.4% (mean 94.5%) of the applied radioactivity. The recovery from the dark control sample was 94.2%. Only one duplicate is
reported due to a spill during transferral of the sample.

VOLATILE DEGRADATION PRODUCTS
The total volatiles evolved for the final time point was equivalent to 3.8% of the applied radioactivity. This was not significant and no further characterisation was carried out.

RADIOACTIVE RESIDUES
- Irradiated samples: The aqueous samples were analysed by HPLC. The results demonstrated that the applied radiolabelled test substance was rapidly degraded in the irradiated samples, with 15.8% parent compound remaining after 3 days of continuous irradiation. For quantification purposes, the amounts of parent compound remaining in the aqueous samples were based on the HPLC analyses using condition (a).
- Dark controls: No significant degradation was observed in the dark control samples (94.1% of the radioactivity was the test substance at the end of the incubation).

DEGRADATION PRODUCTS
Transformation product #1 was the major degradate, reaching a level of 23.0% of applied after 3 days irradiation. Transformation product #2 was also detected but only reached a level of 4.3% of applied radioactivity after 3 days irradiation. In addition, up to 8 minor degradates were resolved. Further HPLC analysis, using a different ion-pairing reagent with a C8 column (condition b), enabled analysis by LC-MS-MS. Product ion spectra were obtained on the authenticated reference standards using electrospray in positive ion mode: (HPLC condition b). Data-dependent tandem mass spectrometry operating in a full MS scan range of 50 - 250 Da was used to identify the parent and related compounds in the 2 DAT sample, by applying a collision energy of 25eV to the most abundant MS ions above a pre-determined threshold value to provide corresponding MS ions (product ion spectra). A minor component was also detected and found to consist of a mixture of transformation products #3 and #4. However, the significant polar unknown component (representing a maximum of 12.2% of the applied radioactivity at the 2 DAT sampling point) could not be identified using HPLC method (b) due to interference from the ion-pairing reagent. Therefore, another method was used with a C18 column and a simple formic acid mobile phase (condition c). The general resolution of this method was not good however the polar unknown component was easily resolved with an improved retention time. Data-dependent tandem mass spectrometry operating in a full MS scan range of 50 - 250 Da and 50 - 500 Da confirmed the presence of the above compounds. The product ion spectrum obtained for the unknown was found to be similar to that produced by transformation product #1, both having 78, 106 and 149 fragment ions. In order to elucidate this unknown component, a representative sample was prepared for analysis by LC-NMR. The region of interest was isolated during LC-NMR then submitted for confirmatory LC-MS-MS. These investigations unambiguously demonstrated the structure to be transformation product #5.

PHOTOLYTIC D50 VALUE
The percentage of applied radioactivity present as parent cation was plotted against hours of irradiation and fitted to simple first order kinetic. For this experiment, the photolytic process follows first order kinetics. The DT50 was estimated to be approximately 31 hours of continuous irradiation, which equivalent to approximately 6.5 days of Tokyo spring sunlight or approximately 2.0 days of summer sunlight (calculated for latitudes of 30, 40 and 50 °N).

Table 1. Mass balance

HAT^a	Replicate	% Aqueous	%CO2	Total
0^b	A	94.8	0.0	94.8
	B	94.4	0.0	94.4
2	A	96.4	0.0	96.4
	B	95.0	0.0	95.0
4.2	A	88.4	0.0	88.4
	B	96.2	0.0	96.2
12	A	93.0	0.2	93.2
	B	92.7	0.2	92.9
16.8	A	90.5	0.3	90.8
	B	93.6	0.3	93.9
25.3	A	99.9	0.5	100.4
	B	99.8	0.5	100.3
47.4	A	90.8	2.1	92.9
	B	91.7	2.1	93.8
72	A	89.3	3.8	93.1
	B	91.6	3.8	95.4
3 DAT Dark control	A^c	94.1	0.0	94.1

a - Hours after treatment (continuous irradiation)

b - Zero time from 3rd treatment regime

c - Only A replicate reported due to small spill of replicate B.

 

Table 2.Summary of the Characterisation / Identification of the Aqueous Samples

(All figures are quoted as % of Applied Radioactivity and are calculated means for the duplicate samples)

HAT^a	% The test substance^b	% #1	% #2	% #5
0	90.9	0	0	0
2	88.1	0	0	0
4	81.4	3.5	0	0
12	60.2	10.7	1.9	5.3
18	58.1	11.9	2.1	4.5
24	61.7	11.1	2.2	7.1
48	32.0	18.6	3.4	12.1
72*	15.8	23.0	4.3	11.4
3 DAT dark control	94.2	0	0	0

a - Hours, of continuous irradiation, after treatment.

b - Purity of test material in the aqueous as determined by HPLC using condition a

* - Data was taken from B duplicate only as "worst case scenario"

Validity criteria fulfilled:

not specified

Conclusions:

The estimated half-life was 31 hours of continuous irradiation, which was equivalent to approximately 6.5 days of Tokyo spring sunlight (which is equivalent to approximately 2 days of summer sunlight at a latitude of 50°N).
The mean mass balance was 94.5% of the applied radioactivity. Mineralisation was also observed, with 14CO2 representing 3.8% of the applied radioactivity at the end of the irradiation period. No significant degradation was observed in the dark control. The most significant known metabolite formed was transformation product #1, reaching a level of 23.0 % after 3 days continuous irradiation. Transformation product #2 was also detected at much lower levels, reaching 4.3 % after 3 days continuous irradiation. Another minor component was elucidated during LC-MSMS, the proposed identification is a mixture of transformation products #3 and #4. The majority of the remainder consisted of a polar component (representing a maximum of 12.1% of the applied radioactivity at the 2 DAT sampling point). Investigation of this component by LC-NMR and confirmation by LC-MS unambiguously demonstrated this compound to be transformation product #5.

Executive summary:

The photolysis of the test substance was investigated in sterile, natural water (Middle Row Pond). Radiolabelled test substance was applied, at a nominal concentration of 10 µg/mL, to the natural water in individual photolysis vessels. The treated solutions were continuously irradiated using light from a xenon arc lamp, which was filtered to give a spectral distribution close to that of natural sunlight. The samples were maintained at 25 ± 2°C and were irradiated for periods up to the equivalent of ca 15 days Tokyo Spring sunlight (which is equivalent to ca 5 days of summer sunlight at latitude of 50°N). This was equivalent to more than two half-lives. Duplicate samples were taken for analysis at 7 intervals during irradiation. Duplicate ‘dark control’ samples were prepared and maintained at 25±2°C. These were analysed at an interval equivalent to that of the longest irradiation to demonstrate that any degradation in the irradiated samples was due to photolysis. The mean mass balance (across all samples) was 94.5% of the applied radioactivity. Trapped volatiles (radiolabelled CO2) accounted for up to 3.8% during the maximum irradiation period. The results demonstrated that the applied radiolabelled test substance was rapidly degraded in the irradiated samples, with 15.8% parent compound detected after 3 days of continuous irradiation. The photo-degradation of the substance followed first order kinetics. The estimated half-life was 31 hours of continuous irradiation, which was equivalent to approximately 6.5 days of Tokyo spring sunlight (which is equivalent to approximately 2 days of summer sunlight at a latitude of 50 °N). A number of photodegradates were formed: in addition to transformation products #1 and #2, eight other discrete peaks were resolved by HPLC of 2 and 3 DAT samples. The most significant known metabolite formed was transformation product #1, reaching a level of 23.0 % in the last sampling point. Traces of transformation product #2 were also detected by HPLC, reaching a maximum level of 4.3% of applied radioactivity after 3 days continuous irradiation. A mixture of transformation products #3 and #4 was detected by LC-MS-MS analysis, however this could not be related back to the quantifiable data. The majority of the remainder consisted of a polar component (representing a maximum of 12.1% of the applied radioactivity at the 2 DAT sampling point), which was unambiguously identified as transformation product #5. No significant degradation was apparent in the ‘dark controls’ indicating that the degradation in irradiated samples was due to photodegradation only.  

Description of key information

DT50 = 2 days (summer sunlight 50 °N), Sterilised natural river water (pH = 7.9), continuous irradiation with Xenon arc lamp for 3 days (39.4 W.m-2 between 300 - 400 nm), 25 °C, OECD TG draft, Oliver & Webb 2005

Key value for chemical safety assessment

Half-life in water:

2 d

Additional information

The photolysis of the test substance was investigated in sterile, natural water (Middle Row Pond). Radiolabelled test substance was applied, at a nominal concentration of 10 µg/mL, to the natural water in individual photolysis vessels. The treated solutions were continuously irradiated using light from a xenon arc lamp, which was filtered to give a spectral distribution close to that of natural sunlight. The samples were maintained at 25 ± 2°C and were irradiated for periods up to the equivalent of ca 15 days Tokyo Spring sunlight (which is equivalent to ca 5 days of summer sunlight at latitude of 50°N). This was equivalent to more than two half-lives. Duplicate samples were taken for analysis at 7 intervals during irradiation. Duplicate ‘dark control’ samples were prepared and maintained at 25±2°C. These were analysed at an interval equivalent to that of the longest irradiation to demonstrate that any degradation in the irradiated samples was due to photolysis. The mean mass balance (across all samples) was 94.5% of the applied radioactivity. Trapped volatiles (radiolabelled CO2) accounted for up to 3.8% during the maximum irradiation period. The results demonstrated that the applied radiolabelled test substance was rapidly degraded in the irradiated samples, with 15.8% parent compound detected after 3 days of continuous irradiation. The photo-degradation of the substance followed first order kinetics. The estimated half-life was 31 hours of continuous irradiation, which was equivalent to approximately 6.5 days of Tokyo spring sunlight (which is equivalent to approximately 2 days of summer sunlight at a latitude of 50 °N). A number of photodegradates were formed: in addition to transformation products #1 and #2, eight other discrete peaks were resolved by HPLC of 2 and 3 DAT samples. The most significant known metabolite formed was transformation product #1, reaching a level of 23.0 % in the last sampling point. Traces of transformation product #2 were also detected by HPLC, reaching a maximum level of 4.3% of applied radioactivity after 3 days continuous irradiation. A mixture of transformation products #3 and #4 was detected by LC-MS-MS analysis, however this could not be related back to the quantifiable data. The majority of the remainder consisted of a polar component (representing a maximum of 12.1% of the applied radioactivity at the 2 DAT sampling point), which was unambiguously identified as transformation product #5. No significant degradation was apparent in the ‘dark controls’ indicating that the degradation in irradiated samples was due to photodegradation only.