4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2016 to 20 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

The study was conducted using two water-sediment systems, Dyfi Estuary (river) and Calwich Abbey Lake (pond).
The sampling locations were not in locations subjected to effluent discharges.

Details on source and properties of sediment:

The sampling was performed in accordance to draft ISO Guidance on sampling of bottom sediments.

Duration of test (contact time):

>= 146 - <= 148 d

Initial conc.:

49.96 µg/L

Based on:

test mat.

Remarks:

MP label

Initial conc.:

49.97 µg/L

Based on:

test mat.

Remarks:

HP label

Initial conc.:

53.32 µg/L

Based on:

test mat.

Remarks:

Oxo label

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

TOC removal

Details on study design:

- Preparation of test system: One-litre all-glass metabolism flasks (inner diameter: approximately 10.6 cm) were filled with sediment to reach a height of approximately 2.7 cm for pond and 2.4 cm for river system. Thereafter, the corresponding water was added to reach a sediment/water volume ratio of approximately 1:3 (173.3 g dry river sediment and 69.6 g dry pond sediment : 700 mL water). Following the start of acclimation, the flasks were ventilated with moistened air. After application of the test item, the flasks were connected to a series of three volatiles traps, the first trap containing ethylene glycol and the second and third traps 2M NaOH (50 mL each) with a polyurethane trap before the trapping flasks. For sampling on day 0, no absorption traps were set up.

- Acclimation: The sediment and water were left to settle in the flasks (= start of the acclimation period); then the samples were ventilated with moistened air. Aeration of the water layer, without disturbing the sediment, was achieved by directing the incoming air carefully into the surface
water layer using a glass capillary.
The samples were left to acclimatise for about 3 weeks at 21 ± 1 °C in the dark before treatment, in order to achieve the most constant redox potential and oxygen conditions possible.

SAMPLING
- Separate surrogate samples treated with the unlabelled test item, along with samples treated with solvent only were taken for parameter measurements. In addition, control samples treated with solvent only were taken to determine the microbial biomass at the start and end of incubation.
- Traps: Traps were used to collect CO2 and volatile organics. Radioactivity in the absorption traps was determined by LSC at the corresponding sampling intervals. Prior to the determination of radioactivity, the volume of the liquid in each sodium hydroxide and ethylene glycol trap was recorded. In order to confirm the presence of 14CO2, the radioactivity in the sodium hydroxide traps was precipitated with barium hydroxide for selected samples. For this purpose, combined aliquots of trapping solution were used after 120 or 121 days for MP- and HP-treated samples and after 53 to 67 days for the OXO-treated samples. Absorption traps were exchanged when necessary.

Test performance:

The test performed as expected and required by the test guidelines.

Compartment:

natural water / sediment: freshwater

% Recovery:

97.2

St. dev.:

1.7

Remarks on result:

other: Overall recovery for applied radioactivity in river system after 146 days (MP label)

Compartment:

natural water / sediment: freshwater

% Recovery:

96.7

St. dev.:

2

Remarks on result:

other: Overall recovery of applied radioactivity in pond system after 146 days (MP label)

Compartment:

natural water / sediment: freshwater

% Recovery:

99.9

St. dev.:

1.5

Remarks on result:

other: Overall recovery of applied radioactivity in river system after 148 days (HP label)

Compartment:

natural water / sediment: freshwater

% Recovery:

99.6

St. dev.:

1.8

Remarks on result:

other: Overall recovery of applied radioactivity in pond system after 148 days (HP label)

Compartment:

natural water / sediment: freshwater

% Recovery:

98.4

St. dev.:

2.8

Remarks on result:

other: Overall recovery of applied radioactivity in river system after 147 days (Oxo label)

Compartment:

natural water / sediment: freshwater

% Recovery:

98.5

St. dev.:

1.8

Remarks on result:

other: Overall recovery of applied radioactivity in pond system after 147 days (Oxo label)

Key result

Compartment:

natural water / sediment: freshwater

DT50:

54.2 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Degradation in total river system

Key result

Compartment:

natural water / sediment: freshwater

DT50:

11.3 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Degradation in total pond system

Transformation products:

yes

No.:

#14

No.:

#13

No.:

#12

No.:

#11

No.:

#10

No.:

#9

No.:

#8

No.:

#7

No.:

#6

No.:

#5

No.:

#4

No.:

#3

No.:

#2

No.:

#1

Volatile metabolites:

yes

Residues:

yes

Details on results:

MASS BALANCE
- The mass balance from all individual soil samples ranged from 90.1% to 104.4%

VOLATILE DEGRADATION PRODUCTS
- Small amounts of radioactivity in the form of organic volatile products (≤ 0.6% AR) were recovered in the ethylene glycol or polyurethane traps. Evolution of 14CO2 increased throughout the study in samples treated with MP Label (max. 19.1% AR) and OXO Label (max. 35.3% AR). 14CO2 did not exceed 0.9% AR in samples treated with HP Label.

DISTRIBUTION OF RADIOACTIVE RESIDUES
- Radioactive residues in water phase: The mean amount of radioactivity in the water phase samples of the river system ranged from 54.3% to 78.0% AR at 0 DAT and from 4.8% to 13.7% AR at the end of incubation. For the pond system, corresponding values ranged from 51.8% to 76.3% AR at 0 DAT and from 8.0% to 12.1% AR at the end of incubation.
- Radioactive residues in sediment extracts: In the river system, total mean extractable radioactivity (including non-polar extractions) ranged from 16.2% AR to 42.8% AR at 0 DAT and from 43.2% to 76.6% AR at the end of incubation. Corresponding values for pond system samples ranged from 19.8% AR to 44.7% AR at 0 DAT and from 46.6% to 79.4% AR at the end of incubation. The majority of the radioactivity was extracted with acetonitrile/water, with much smaller amounts being extracted in the THF/hexane extracts. Mean radioactivity from extractions with THF ranged from 0.8% to 2.3% AR at 0 DAT and from 3.1% to 5.6% AR at the end of incubation in the river system. In the pond system, mean radioactivity from extractions with THF ranged from 1.2% to 2.4% AR at 0 DAT and from 3.1% to 4.8% AR at the end of incubation. Contributions from THF extracts remained less than 6% AR at all sampling intervals for both systems. Maximum mean contributions from isohexane extracts were 1.2% for the river system and 1.4% AR for the pond system.
- Non-extractable radioactivity: The mean amount of non-extractable radioactivity in the river system was ≤ 0.1% at 0 DAT and ranged from 10.8% to 16.7% AR at the end of incubation. In the pond system, the mean amount of non-extractable radioactivity ranged from 0.1% to 0.2% AR and from 9.8% to 18.5% AR at the end of incubation.

DEGRADATION OF THE TEST ITEM AND PATTERN OF METABOLITE FORMATION
- The amount of test item in the total river test system decreased from a mean (across all 3 labels) of 94.1% AR at 0 DAT to a mean of 30.6% (across all 3 labels) AR by the end of the incubation period. In the total pond system, the amount of test item decreased from a mean (across all 3 labels) of 93.6% AR at 0 DAT to a mean of 2.1% AR (across all 3 labels) by the end of the incubation period. In both systems, 14C-labelled test substance was present in the water phase as well as the sediment extracts, slightly predominating in the sediment. The test substance was present in the water phase just after application, but dissipated rapidly in both systems thereafter. Likewise the test substance was present in the sediment extracts just after application and its amount increased until 2 to 8 DAT to a maximum and decreased thereafter-
In addition to the applied test item, the test substance, 14 metabolites were identified. In total, there were seven metabolites present at ≥ 10% AR, namely:
- M2 present in both the water and sediment phases of the river system and in the sediment phase of the pond system at up to a maximum mean of 10.5% AR (Dyfi Estuary, HP label, 148 DAT) in the total system.
- M4 was present in both the water and sediment phases at up to a maximum mean of 18.1% AR (Calwich Abbey, MP label, 8 DAT) in the total system and declined thereafter to <1% AR by 100 DAT.
- M5 was present in both the water and sediment phases at up to a maximum mean of 20.4% AR (Calwich Abbey, MP label, 5 DAT) in the total system and thereafter declined to undetectable levels by the end of the study.
- M7 was present mainly in the sediment phase at up to a maximum mean of 30.0% AR (Calwich Abbey, HP label, 100 DAT) in the total system, and declined slightly by 148 DAT.
- M8 was present in both the water and sediment phases at up to a maximum mean of 28.9% AR (Calwich Abbey, MP label, 13 DAT) in the total system and declined slowly thereafter to 15.1% AR in that system treated with MP label by the end of the study.
- M13 was present in the water phase only at up to a maximum mean of 12.6% AR (Calwich Abbey, MP label, 27 DAT) in the total system and declined thereafter to 1.6% AR in that system treated with MP label by the end of the study.
- M6 and M15 was present in both the water and sediment phases at up to a maximum mean of 30.3% AR (Calwich Abbey, HP label, 100 DAT).
In addition to the metabolites present at ≥ 10% AR, four additional metabolites were present
at levels between 5 and 10% AR, namely:
- M1 was present in both the water and sediment phases at up to a maximum mean of 7.9% AR (Calwich Abbey, MP label, 146 DAT) in the total system.
- M49was present in the sediment phase only at up to a maximum mean of 8.1% AR (Dyfi Estuary, HP label, 148 DAT) in the total system.
- M46 was present in both the water and sediment phases at up to a maximum mean of 7.5% AR (Calwich Abbey, MP label, 146 DAT) in the total system.
- M14 was present at up to a maximum mean of 6.0% AR (Dyfi Estuary,OXO label, 13 DAT) in the total system and declined thereafter to 2.0% AR by the end of the incubation period. This metabolite was only present at > 5% at a single time point and was found in both the water and sediment phases.
Three further identified fractions were present at levels < 5% AR, namely:
- M10 (River [MP, HP and OXO Labels], Pond [HP Label]) reached a maximum level of 4.3% AR in the river system.
- M16 (River and Pond [MP and OXO Label]) reached a maximum of 4.1% AR in the river system.
- M28 (River and Pond [HP Label]) reached a maximum of 4.6% AR in the river system.
The identity of all metabolites present at >5% AR was confirmed by HPLC-MS and by co-chromatography with an authentic reference standard by HPLC and 2D-TLC. Good agreement between HPLC and TLC results were observed.

RATE OF DISSIPATION AND DEGRADATION ON 14C-LABELLED TEST SUBSTANCE
The rate of degradation of 14C-labelled test substance, in each of the water-sediment total systems, and the rate of dissipation from each respective individual water phase were estimated using Single First-Order (SFO) reaction kinetics. DegT50/DT50 for the total system of the river and pond systems were 54.2 and 11.3 days, respectively, with DegT50/DT50 values of 4.16 and 2.29 days for the individual water phases of the river and pond systems, respectively. DegT90/DT90 for the total system of the river and pond systems were 180 and 37.4 days, respectively, with DegT90/DT90 values of 13.8 and 7.6 days for the individual water phases of the river and pond systems, respectively.

PROPOSED DEGRADATION PATHWAY
The data from this study show that the test substance is degraded in aerobic water-sediment systems by opening of the isoxazoline and oxoisoxazolidine rings via reduction and hydrolysis. In addition, some mineralisation of the ring systems is evident by the generation of 14CO2. Formation of significant amounts of bound residues was observed towards the end of the incubation period.

Conclusions:

The DT50 of test substance in the total aquatic systems was determined to be 54.2 days and 11.3 days in the river and pond systems, respectively. The test substance dissipated from the water phases of the river and pond systems, with a DT50 of 4.16 days and 2.29 days, respectively.

Executive summary:

The route and rate of degradation of 14C-labelled test substance (labelled in three positions) was investigated under GLP in two different aquatic sediment systems: a river (Dyfi Estuary, UK) and a pond (Calwich Abbey Lake, UK) to OECD TG 308. 14C-labelled test substance was applied at a rate of 49.96 µg/L (Methylphenyl [MP] Label), 49.97 µg/L (Halophenyl [HP] Label) and 53.32 µg/L (Oxoisoxazolidinyl [OXO] Label). The water/sediment for both the river and pond systems was incubated under aerobic conditions in the laboratory in the dark at a temperature of 21 ± 1 °C for up to 148 days. Treated samples were continuously ventilated with moistened air and the outlet air was passed through a trapping system consisting of polyurethane foam, a flask with ethylene glycol and two flasks with sodium hydroxide in series. Samples were taken regularly during the incubation, and the water phase was separated from the sediment phase and analysed by LSC. The sediment samples were exhaustively extracted using solvents of varying polarity. The radioactivity in the extracts was measured by LSC. The water phase and the extractable 14C-residues from the sediment were characterised by HPLC. Selected water phases and pooled extracts were also analysed by two-dimensional thin-layer chromatography (2D-TLC) and liquid chromatography with tandem mass spectrometry (LCMS/MS) to confirm the identity of radioactive components. Any volatile radioactivity was continuously flushed from the vessels and collected in traps and analysed. Remaining unextracted residues were analysed by combustion. A mass balance was determined for each sample.
The degradation rate of the parent test item in the water and in the total water-sediment system was calculated according to single first-order (SFO) kinetics.
For river test system samples, mass balance values in individual samples ranged from 90.1% to 104.4% applied radioactivity (AR). For the pond test system samples, individual mass balance values ranged from 90.9% to 102.3% AR. The mean amount of radioactivity in the water phase samples of the river system ranged from 54.3% to 78.0% AR at 0 DAT and from 4.8% to 13.7% AR at the end of incubation. For the pond system, corresponding values ranged from 51.8% to 76.3% AR at 0 DAT and from 8.0% to 12.1% AR at the end of incubation.

In the river system, total mean extractable radioactivity ranged from 16.2% AR to 42.8% AR at 0 DAT and from 43.2% to 76.6% AR at the end of incubation. Corresponding values for pond system samples ranged from 19.8% AR to 44.7% AR at 0 DAT and from 46.6% to 79.4% AR at the end of incubation. The mean amount of non-extractable radioactivity in the river system was ≤ 0.1% at 0 DAT

and ranged from 10.8% to 16.7% AR at the end of incubation. In the pond system, the mean amount of non-extractable radioactivity ranged from 0.1% to 0.2% AR and from 9.8% to 18.5% AR at the end of incubation. In the river system, evolution of 14CO2 increased throughout the study in samples treated with MP Label (max. 16.6% AR at the end of incubation) and OXO Label (mean max. 35.3% AR at the end of incubation). Mean 14CO2 amounts did not exceed 0.9% AR in samples treated with HP Label. Radioactivity in the form of organic volatile products did not exceed 0.4% AR in the ethylene glycol or polyurethane traps. A similar pattern was observed in the pond

system. 14CO2 increased throughout the study in samples treated with MP Label (mean max. 19.1% AR) and OXO Label (mean max. 27.2% AR). Mean 14CO2 did not exceed 0.1% AR in individual samples treated with HP Label. Radioactivity in the form of organic volatile products did not exceed 0.6% AR in the ethylene glycol or polyurethane traps.

The amount of test item in the total river test system decreased gradually from a mean (across all 3 labels) of 94.1% AR at 0 DAT to a mean (across all 3 labels) of 30.6% AR by the end of the incubation period. In the total pond system, the amount of test item decreased from a mean (across all 3 labels) of 93.6% AR at 0 DAT to a mean (across all 3 labels) of 2.1% AR by the end of the incubation period. In both systems, 14C-labelled test substance was present in the water phase as well in the sediment extracts, slightly predominating in the sediment.

In addition to applied test item, 14 metabolites were identified. In total, there were seven metabolites present at ≥10% AR in the total water-sediment systems: M2 present up to a mean maximum of 10.5% AR in the total system (River [HP Label]); M4 present up 18.1% AR in the total system (Pond [MP Label]); M5 present up to 20.4% AR (Pond [MP Label]); M7 present up to 30.0% AR (Pond [HP Label]); M8 present up to 28.9% AR (Pond [MP Label]); M13 present up to 12.6% AR (Pond [MP Label]) and M6/M15 present up to 30.3% AR (Pond [HP Label]). In addition to the metabolites present at ≥10% AR, four additional metabolites were present at levels between 5 and 10% AR, namely: M1 (River and Pond (MP Label), Pond [HP Label]); M49 (River [MP Label and HP Label]); M46 (Pond [MP Label and HP Label]) and M14 (River [OXO Label; present at > 5% at a single time point]). Three further identified fractions were present at levels <5% AR, namely: M10 (River [MP, HP and OXO Labels], Pond [HP Label]); M16 (River and Pond [MP and OXO Label]) and M28 (River and Pond [HP Label]). No other unassigned metabolite present in chromatographed fractions exceeded 5% AR. The data from this study show that the test substance was degraded in aerobic water-sediment systems by opening of the isoxazoline and oxoisoxazolidine rings via reduction and hydrolysis to afford a major hydroxy-ketone metabolite M8 via metabolites M4 and M5 and minor metabolites e.g. M14. Reduction of the ketone moiety in M8 to afford metabolite M6/M15. Cleavage of the hydroxy-ketone metabolites to afford M28, M16 and M13. Reduction of the ketone moiety resulting from cleavage of the hydroxy-keto metabolites to afford a major metabolite metabolite M7. Hydrolysis of the amide moiety to afford metabolites M1, M2 and M46. Further breakdown and/or loss of the oxoisoxazolidine ring to afford the amide metabolite M49. In addition, some mineralisation of the ring systems as is evident by the generation of 14CO2. Formation of significant amounts of bound residues was observed towards the end of the incubation period.
The DT50 of test substance in the total aquatic systems was determined to be 54.2 days and 11.3 days in the river and pond systems, respectively. The test substance dissipated from the water phases of both the river and pond systems, with a DT50 of 4.16 days and 2.29 days, respectively.

Description of key information

Freshwater sediment, DT50 = 54.2 d, 21 °C, OECD TG 308, Völkel 2019

 

Key value for chemical safety assessment

Half-life in freshwater sediment:

54.2 d

at the temperature of:

21 °C

Additional information

The route and rate of degradation of 14C-labelled test substance was investigated under GLP to OECD TG 308 in two aquatic sediment systems: a river (Dyfi Estuary, UK) and a pond (Calwich Abbey Lake, UK). The test substance was labelled at three distinct positions, a methylphenyl (MP), a halophenyl (HP) and the oxoisoxazolidinyl (OXO) position. The applied test concentration was about 50 µg/L for each labelled test substance. The water/sediment test systems were incubated under aerobic laboratory conditions in the dark at a temperature of circa 21 °C for up to 148 days. Duplicate samples were taken for analysis at different time points of incubation. For each sample, the water phase was separated from the sediment phase to be analysed by liquid scintillation. The sediment samples were exhaustively extracted using solvents of varying polarity and extracts were also analysed by liquid scintillation or by HPLC.

For river test system samples, mass balance values in individual samples ranged from 90.1% to 104.4% applied radioactivity (AR). For the pond test system samples, individual mass balance values ranged from 90.9% to 102.3% AR. The mean amount of radioactivity in the water phase samples of the river system ranged from 54.3% to 78.0% AR at study begin and from 4.8% to 13.7% AR at the end of incubation. For the pond system, corresponding values ranged from 51.8% to 76.3% AR at study begin and from 8.0% to 12.1% AR at the end of incubation.

In the river system, total mean extractable radioactivity in sediment ranged from 16.2% AR to 42.8% AR at the start and from 43.2% to 76.6% AR at the end of incubation. Corresponding values for pond system samples ranged from 19.8% AR to 44.7% AR at study begin and from 46.6% to 79.4% AR at the end of incubation.

The mean amount of non-extractable radioactivity in the river system was ≤ 0.1% at the begin and ranged from 10.8% to 16.7% AR at the end of incubation. In the pond system, the mean amount of non-extractable radioactivity ranged from 0.1% to 0.2% AR at the start and from 9.8% to 18.5% AR at the end of incubation. In the river system, evolution of 14CO2 increased throughout the study in samples treated with MP Label (max. 16.6% AR at the end of incubation) and OXO Label (mean max. 35.3% AR at the end of incubation). Mean 14CO2 amounts did not exceed 0.9% AR in samples treated with HP Label. Radioactivity in the form of organic volatile products did not exceed 0.4% AR in the ethylene glycol or polyurethane traps. A similar pattern was observed in the pond system. 14CO2 increased throughout the study in samples treated with MP Label (mean max. 19.1% AR) and OXO Label (mean max. 27.2% AR). Mean 14CO2 did not exceed 0.1% AR in individual samples treated with HP Label. Radioactivity in the form of organic volatile products did not exceed 0.6% AR in the ethylene glycol or polyurethane traps.

The amount of test substance in the total river system decreased gradually from a mean of 94.1% applied radioactivity (AR) at study begin to a mean of 30.6% AR at the end of the incubation period. The corresponding numbers in the pond system were 93.6% at study begin and 2.1% at the end of the incubation period. In both systems, 14C-labelled test substance was present in the water phase as well in the sediment extracts, slightly predominating in the sediment. The data from this study show that the test substance is degraded in aerobic water-sediment systems by opening of the isoxazoline and oxoisoxazolidine rings via reduction and hydrolysis. In addition, some mineralisation of the ring systems is evident by the generation of 14CO2. Formation of significant amounts of bound residues was observed towards the end of the incubation period.

Based on the findings, the DT50 of test substance in the total aquatic systems was determined to be 54.2 days and 11.3 days in the river and pond systems, respectively. The test substance dissipated in the water phases of both the river and pond systems, with a DT50 of 4.16 days and 2.29 days, respectively.