ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Subdivision N Pesticide Guideline 162-3 (Anaerobic Aquatic Metabolism)

Qualifier:

equivalent or similar to guideline

Guideline:

other: Food Production and Inspection Branch, Agriculture Canada (1987) Part-1: Soil (Laboratory) - Degradation Pathways in Chapter 2: Laboratory Studies, Section C: Biotransformation of Environmental Chemistry and Fate Guidelines for Reg. of Pesticides T-1- 255

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

anaerobic

Remarks:

2 flasks were flushed with humidified air beginning on day 120 to check whether degradation would increase in aerobic conditions

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

- Details on collection (location): Geographic location: pond, AgrEVO Research Centre, Pikeville, North Carolina, USA
- Temperature (°C) at time of collection:
- pH at time of collection: 6.4
- Electrical conductivity: 0.07 mS/cm
- Hardness (CaCO3): 40 mg/L
- Water filtered: no

Details on source and properties of sediment:

- Details on collection (location, procedure): Geographic location: pond, AgrEVO Research Centre, Pikeville, North Carolina, USA. The sediment was collected from below the surface of the water (depth 15-100cm) to a depth of approximately 5 to 30 cm from the water/sediment interface.
- Textural classification (USDA %sand/silt/clay): 39 / 39.8 / 21.2
- pH at time of collection: 5.6
- Organic carbon (%): 2.91
- CEC (meq/100 g): 9.47
- Bulk density (g/cm³): 1.12
- Sediment samples sieved: yes, 2 mm sieve

Duration of test (contact time):

365 d

Initial conc.:

61.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution used/treatment: Using the equivalent maximum field application rate of 150 g/ha (0.134 Ib/acre) and an internal diameter of 7.2 cm of the test system flask (area = 40.72 cm2) for the surface of the water, the application rate translated to 61.1 ug AE F122006 per flask. A solution of [U-14C-diphenyl]-AE F122006 was prepared in acetone and the concentration calculated by liquid scintillation counting. Aliquots of test solution (140 uL) were carefully applied to the water surface of each flask.
- Test temperature: Prepared flasks were placed in a dark environmental chamber set to maintain 20 ± 1 °C. A computer data-logging system was used to record the temperature every hour. The actual mean temperature over the 398 day incubation and experiment period was 19.87 ± 0.43 °C (mean ± standard deviation). There were episodes of personnel entering/exiting the chamber and some environmental chamber malfunctions that caused the temperature to be outside the 20 ± 1 °C range. These excursions totaled 0.6% of the 398 day period and were not considered to affect the experiment adversely.
- pH adjusted: no
- Continuous darkness: yes/no

TEST SYSTEM
- Culturing apparatus: Sediment (75 mL) and pond water (225 mL) were combined in flasks which were fitted with a bubbler tube (located 1-2 cm below the water surface) which supplied a steady stream of humidified nitrogen. Nitrogen flow was regulated by a uniform capillary flow cell. Each flask was flushed with nitrogen that had been humidified by being continuously passed through deionized water prior to entering the treatment flask. The flasks were placed in a dark environmental chamber at 20 ± 1 °C for 33 days prior to treatment to establish anaerobic conditions before addition of test substance.
- Method used to create anaerobic conditions: See nitrogen bubbler under "Culturing apparatus"; Nitrogen flow through the flow-through test system was regulated by a uniform capillary flow cell. Humidified nitrogen was passed through each flask continuously. Eh ranges for aerobic microbial respiration have been reported to be between approximately 200 mV to 850 mV
- Details of traps for CO2 and organic volatile, if any: After exiting the test flask, the nitrogen passed through an ethylene glycol trap to collect organic volatiles and an ethanolamine trap to collect carbon dioxide. Trap saturation was monitored in some instances by adding a second ethanolamine trap. The traps in the test system were interconnected with glass and Tygon® tubing in such a way as to minimize exposure of the nitrogen stream to Tygon® tubing. The effluent gas was passed through traps containing ethylene glycol and ethanolamine for the collection of volatile organic components and carbon dioxide, respectively.

SAMPLING
- Sampling frequency: Days 0, 7, 14, 30, 61, 120, 181, 273, and 365.
- Sampling method:
- Sterility check if applicable: sampled on days 7, 14, 30
- Sample storage before analysis: All samples were processed through ambient extraction on the day of harvest. Extracts were stored at approximately -20 °C. Analytical chromatography was carried out within 41 days of sampling, with the majority being analyzed within 14 days. Stability for over 9 months was demonstrated by repeat HPLC analysis of representative fractions. Extracted sediments were air-dried and stored at -20 °C prior to combustion analysis. Recovery of radioactivity was good with an average of 95.3% over the whole study.

Compartment:

other: water, material (mass) balance

% Recovery:

29.7

Compartment:

other: sediment, material (mass) balance

% Recovery:

11.3

Compartment:

entire system

DT50:

0.6 d

Type:

(pseudo-)first order (= half-life)

Compartment:

entire system

DT50:

1.3 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: recalculated from 20°C to 12°C

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Formation and decline of each transformation product during test: Through rapid hydrolysis, ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate degraded to 5,5-diphenyl-2-isoxazoline-3-carboxylic acid with a DT50 value of 0.6 days. The disposition of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate from the water to sediment phase was insignificant and therefore the process was not included in the kinetic model. 5,5-diphenyl-2-isoxazoline-3-carboxylic acid readily dissipated from the water column with an overall DT50 of 19.5 days. Partitioning of 5,5-diphenyl-2-isoxazoline-3-carboxylic acid from the water phase to the extractable residue in sediment was relatively low. 3-hydroxy-3,3-diphenylpropanenitrile formed in the water phase was slowly adsorbed to the sediment phase and was found relatively stable under anaerobic conditions.
- Description of biotransformation pathway: The primary route of degradation in soil was via hydrolysis to 5,5-diphenyl-2-isoxazoline-3-carboxylic acid, which has also been shown to be the principal aqueous hydrolysis product of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate. Further degradation of 5,5-diphenyl-2-isoxazoline-3-carboxylic acid occurred in this study as indicated by the high amount of mineralization (75.7% after Day 119 in Chantepie soil). No mineralization of 5,5-diphenyl-2-isoxazoline-3-carboxylic acid was observed in sterile soil, where this degradate was persistent. Intermediate transient degradates involving opening of the isoxazole ring of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate were detected. These were the 3-hydroxynitrile 3-hydroxy-3,3-diphenylpropanenitrile and its partial hydrolysis product, the amide 3-hydroxy-3,3-diphenylpropanamide.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

TEST CONDITIONS
- Anaerobicity maintained throughout the study: Yes

STERILE TREATMENTS
- Transformation of the parent compound: Sterile samples were analyzed over the initial 30 days of incubation. During this period, more of the radioactivity remained in the water phase of the sterile samples (65.7%) than in the non-sterile samples (49.7%). The sediment associated residue was, in both cases, readily recovered by solvent extraction leaving only 3-4% non-extractable.
- Formation of transformation products: As in the non-sterile samples, AE F122006 rapidly disappeared from the sterile system. By Day 30, in the sterile system, the acid AE F129431 was the predominant species. The major residue in the non-sterile system was the nitrile AE C637375, found primarily in the sediment. It appears, therefore, that the key mechanisms at work in the non-sterile anaerobic aquatic system are the initial chemical hydrolysis of AE F122006 to AE F129431 followed by primarily biotic transformation of the acid to the nitrile AE C637375.
- Formation of extractable and non-extractable residues: By Day 30, the acid AE F129431 was the predominant species present in both the water and sediment.

The radioactivity detected in volatile traps reached a maximum of only 3.6% (mean of two samples) of the applied radioactivity on Day 365 under anaerobic conditions. However, once nitrogen was replaced by humidified air and aerobic conditions established, up to 20.0% of applied radioactivity was mineralized to carbon dioxide.

Table 1: DT50 and DT90 values for the degradation of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate and its metabolites in the in the water/sediment system at 20 °C

Compound	DT50	DT90
Degradation in Sediment-Water system under anaerobic conditions
ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate	0.6	1.9
5,5-diphenyl-2-isoxazoline-3-carboxylic acid	22.5	74.8
3-hydroxy-3,3-diphenylpropanenitrile	888.7	2952.0
3-hydroxy-3,3-diphenylpropanamide	433.2	1439.1
NER	778.8	2587.2
CO2	n.a.	n.a.
Degradation in Sediment-Water system under aerobic conditions*
3-hydroxy-3,3-diphenylpropanenitrile	81.5	270.9

n.a. = not applicable

* = converted on day 120

Validity criteria fulfilled:

not applicable

Reference 2

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Transformation products:

not measured

Reference 3

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA Subdivision N Pesticide Guideline 162-4 (Aerobic Aquatic Metabolism)

Version / remarks:

United States Environmental Protection Agency (EPA) Pesticide Assessment Guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

other: Canadian Guidelines for Determining Environmental Chemistry and Fate of Pesticides; PMRA: T-1 -255

Qualifier:

equivalent or similar to guideline

Guideline:

other: Society of Environmental Toxicology and Chemistry (SETAC - Europe) Procedures for Assessing the Environmental Fate and Ecotoxicology of Pesticides set forth in European Union Commission Directive:95/36/EEC of 14 July 1995

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

- Details on collection (location, procedure): The Hoechst sediment and overlying water were collected on June 25, 1997 from a gravel pit site in Hoechst, Germany. The North Carolina sediment and overlying pond water were collected from a pond at the AgrEvo Research Center on July 27, 1998 located in Pikeville, North Carolina, USA. The pH, temperature, and dissolved oxygen contents were measured in situ.
- pH at time of collection: Hoechst Batch 1: 7.6, Batch 2: 7.5; North Carolina: 6.1
- Electrical conductivity: Hoechst Batch 1: 0.69, Batch 2: 0.69; North Carolina: 0.09 mS/cm
- Hardness (CaCO3): Hoechst Batch 1: 312, Batch 2: 306; North Carolina: 36 mg CaCO3 / L
- Dissolved organic carbon (%): Hoechst Batch 1: 0.85; North Carolina: 2.35
- Biomass (CFU/mL): Hoechst : 9.0E+07; North Carolina: 2.4E+05
- Water filtered: no

Details on source and properties of sediment:

- Details on collection (location, procedure): The Hoechst sediment and overlying water were collected on June 25, 1997 from a gravel pit site in Hoechst, Germany. An additional batch of the Hoechst sediment was collected from the same location on August 13, 1998 in order to repeat timepoints up to 30 hours. The North Carolina sediment and overlying pond water were collected from a pond at the AgrEvo Research Center on July 27, 1998 located in Pikeville, North Carolina, USA. Each sediment was collected from below the surface of the water (depth 15-100 cm) to a depth of approximately 5 to 30 cm from the water/sediment interface. The pH, temperature, and dissolved oxygen contents were measured in situ.
- Textural classification (USDA %sand/silt/clay): Hoechst Batch 1: 94/3.2/2.8; Hoechst Batch 2: 98.8 / 0 / 2.4; North Carolina: 48.4 / 29.6 / 22.0
- pH at time of collection: Hoechst Batch 1: 7.8, Batch 2: 7.5; North Carolina: 5.6
- Organic carbon (%): Hoechst Batch 1: 0.85, North Carolina: 2.35
- CEC (meq/100 g): Hoechst Batch 1: 1.46, Batch 2: 0.6; North Carolina: 8.88
- Bulk density (g/cm³): Hoechst Batch 1: 1.40, Batch 2: 1.59; North Carolina: 1.27
- Sediment samples sieved: yes

Duration of test (contact time):

188 d

Initial conc.:

61.1 other: µg per flask

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution used/treatment: 218 µL (Hoechst soil with ESR-99), 139 µL (North Carolina soil), 146 µL (Hoechst soil with ESR-219)
- Test temperature: A temperature data logging system was used to record temperatures every hour. For the Hoechst sediment system, the actual mean temperature over the 189 day incubation and experiment period was 20.2 ± 0.57 °C (mean ± standard deviation, n = 4575). There were
episodes of entering/exiting the chamber and some environmental chamber malfunctions, which caused the temperature to be outside the 20 ± 1 °C range. These excursions totaled 6.3% of the time, with a range of 18.2 - 21.3 °C. For the North Carolina sediment system, the actual mean temperature over the 173 day incubation and experiment period was 19.9 ± 0.29 °C (mean ± standard deviation, n = 4172). There were episodes of entering/exiting the chamber and some environmental chamber malfunctions, which caused the temperature to be outside the 20 ± 1 °C range. These excursions totaled 0.9% of the time, with a range of 17.7 - 25.8 °C. These temperature variations were not considered to adversely affect the experiment.
- pH: Aqueous Buffer = Aqueous 0.01 M Ammonium Acetate, pH 5.8-6.0
- pH adjusted: no

TEST SYSTEM
- Culturing apparatus: Sediment (75 ml) and pond water (225 mL) were combined in flasks and fitted with a bubbler tube (located 1-2 cm below the water surface) which supplied a steady stream (ca. 5-20 mL/min) of humidified atmospheric air regulated by a capillary flow cell.
- Number of culture flasks/concentration: Ten flasks of each system were sterilized and several flasks remained untreated to be used as sterile and non-sterile controls or for retreatment if needed.
- Method used to create aerobic conditions:
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: Traps containing ethylene glycol, ethanolamine and/or sodium hydroxide were used to collect any volatile products generated during the in life phase of the experiment.

SAMPLING
- Sampling frequency: Sampling Hoechst sediment on days 0.04, 0.08, 1, 5, 7, 14, 22, 28, 62, 120, 188 after treatment, Sampling North Carolina sediment on days 0.04, 1, 2, 6, 14, 22, 34, 61, 91, 132.
- Sterility check if applicable: Hoechst sediments: pre-treatment, days 15, 28, 63 after treatment; North Carolina sediments: pre-treatment, days 1, 12, 29, 43 after treatment

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Ten flasks of each system were sterilized

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: 86.8% to 100.3% Hoechst sediment/ water system; 86.5% to 99.9% in the North Carolina system

Compartment:

entire system

DT50:

0.19 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Hoechst system (initial pH = 8)

Compartment:

entire system

DT50:

1.5 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: North Carolina system (initial pH =4.7)

Compartment:

entire system

DT50:

0.4 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: recalculated from 20°C to 12°C

Compartment:

entire system

DT50:

3.2 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: recalculated from 20°C to 12°C

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Formation and decline of each transformation product during test: ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate rapidly degraded to the 5,5-diphenyl-2-isoxazoline-3-carboxylic acid with a DT50 of 0.24 days (about 6 hours) as a consequence of rapid hydrolysis in the water column. 5,5-diphenyl-2-isoxazoline-3-carboxylic acid formed in the water column readily degraded to 3-hydroxy-3,3-diphenylpropanenitrile non-extractable residue (overall DT50 in water column=28 days). Partitioning of 5,5-diphenyl-2-isoxazoline-3-carboxylic acid from the water phase to the extractable residue in sediment was relatively low. 5,5-diphenyl-2-isoxazoline-3-carboxylic acid in the sediment phase desorbed into the water. Almost equal amounts of 3-hydroxy-3,3-diphenylpropanenitrile were detected in the water and sediment phases, both of which were readily degraded to 3-hydroxy-3,3-diphenylpropanamide (DT50 in water phase=28 days) and degraded to a non-extractable residue and 14CC>2. 3-hydroxy-3,3-diphenylpropanamide in the water and sediment phases was detected about 15 days after the dosing of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate and was mineralized to 14CO2 and degraded to a nonextractable residue with a DT5o far less from water of 15 days.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes, but aerobic conditions in the water phase changed to mildly reducing conditions below the sediment surface

STERILE TREATMENTS
- Transformation of the parent compound:
- Formation of transformation products: 5,5-diphenyl-2-isoxazoline-3-carboxylic acid was more persistent in sterile samples but some degradation to 3-hydroxy-3,3-diphenylpropanenitrile was oberved (up to 25% after 62 days). Virtually no 14CO2 was detected in sterile samples, thus, virtually no mineralization occurred.
- Formation of extractable and non-extractable residues: other than the identifiable extractable transformation products, some radioactivity remained in the non-extractable fraction of the sterile samples

The non-extractable residue (NER), reached a maximum of 30.6% in the Hoechst sediment at Day 188. The NER in the North Carolina sediment reached 28.8% of applied radioactivity at Day 22 before the addition of the Soxhlet extraction step, which

reduced the NER to 12.6% at the next timepoint (Day 34). Levels of NER in the North Carolina sediment system did not accumulate significantly, reaching a maximum of 16.4% of applied radioactivity at termination (Day 132). Increasing levels of¹⁴CO₂relative to levels of NER in both sediments suggested that the radioactivity associated with the NER was susceptible to further degradation and

mineralization.

Table 1: DT50 and DT90 values for the dissipation of ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate and its metabolites in the water column of each system and the degradation in the water/sediment system at 20 °C

	Hoechst System DT50                  DT90		North Carolina System DT50                   DT90
Dissipation from Water Phase
Ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate	0.24	0.75	1.2	4.2
5,5-diphenyl-2-isoxazoline-3-carboxylic acid	20	84	12	41
3-hydroxy-3,3-diphenylpropanenitrile	28	97	0.8	25
3-hydroxy-3,3-diphenylpropanamide	15	49	15	49
Degradation in Water + Sediment
Ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate	0.19	0.62	1.5	5.0
5,5-diphenyl-2-isoxazoline-3-carboxylic acid	38	127	21	71
3-hydroxy-3,3-diphenylpropanenitrile	29	97	56	184
3-hydroxy-3,3-diphenylpropanamide	37	124	131	434

Validity criteria fulfilled:

not applicable

Description of key information

DT50 of parent substance in sediment water system: 04 - 3.2 days at 12°C (aerob, US-EPA 162-4, two sediment/water systems).

DT50 of parent substance in sediment water system: 1.3 days at 12°C (anaerob, US-EPA 162-3, one sediment/water system).

Additional information

Two studies investigating the transformation in sediment/water systems under aerobic and anaerobic conditions are available. The key study was performed according to US-EPA guideline 162-4 (GLP). The degradation of radiolabeled parent substance, isoxadifen-ethyl, under aerobic conditions was investigated in two different sediment/water systems. The parent substance was rapidly hydrolysed in both sediment/water systems. The determined DT50 values for the parent compound were 0.4 d and. 3.2 d at 12°C for the entire system. Three metabolites of isoxadifen-ethyl were found with DT50 values of 38 d and 21 d (5,5-diphenyl-2-isoxazoline-3-carboxylic acid at 20 °C), 29 d and 56 d (3-hydroxy-3,3-diphenylpropanenitrile at 20 °C), and 37 d and 131 d (3-hydroxy-3,3-diphenylpropanamide at 20 °C).

Overall, carbon dioxide formation was considerable indicating that mineralisation was a major dissipation pathway of the test item. Formation of carbon dioxide and metabolites demonstrates the complete degradation of the substance in water-sediment systems.

The supporting study was performed according to US-EPA guideline 162-3 (GLP). The degradation of radiolabeled parent substance, isoxadifen-ethyl, under anaerobic conditions was investigated in a sediment/water system. The parent substance was rapidly hydrolysed in the sediment/water system. The determined DT50 values for the parent compound was 1.3 d at 12°C for the entire system. The three metabolites of isoxadifen-ethyl showed DT50 values of 22.5 d ( 5,5-diphenyl-2-isoxazoline-3-carboxylic acid at 20 °C), 888.7 d (3-hydroxy-3,3-diphenylpropanenitrile at 20 °C), and 433.2 d (3-hydroxy-3,3-diphenylpropanamide at 20 °C). Overall, mineralization was negligible under anaerobic conditions. However, when converting the metabolite with the highest DT50 value (3-hydroxy-3,3-diphenylpropanenitrile) to aerobic conditions after 120 days a mineralization to CO2 was determined.