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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Reference
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-09-2018 to 17-05-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Specific details on the test material used for the study are provided in "Test material information" and "Specific details on test material used for this study (confidential)" fields.
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Remarks:
Water and associated sediments from the natural freshwater source (cited in the full study report) were used in the study. Collection 19 September 2018, and were stored in a refrigerator at ca. 4 °C upon delivery to the testing facility
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Water and associated sediments from the Brandywine Creek (Pennsylvania, USA).
- Storage conditions: Collection 19 September 2018, and were stored in a refrigerator at ca. 4 °C upon delivery to the testing facility
- Storage length: Less than 24 hours
- Temperature (°C) at time of collection: 21.4°C
- pH at time of collection: pH 6.92
- Electrical conductivity: Not reported.
- Redox potential (mv) initial/final: Not reported.
- Oxygen concentration (mg/l) initial/final: 7.9 mgO2/L at time of collection ; the ranges of pH and dissolved oxygen content in control samples during the study were 6.60 to 7.27 and 8.45 to 9.07 mg/L, respectively. Aerobic conditions were maintained throughout the study.
- Hardness (CaCO3): Not reported.
- Dissolved organic carbon (%): Not reported. The Organic Carbon content of the filtered water was: 6.819 mg/L
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): See below for prepared combined water/sediment : A standard plate count proved microbial presence (2.0×104 cfu/mL)
- Water filtered: Yes
- Type and size of filter used, if any: Water was filtered through loosely packed glass wool, and the total suspended solids (TSS) concentration and organic carbon concentration of the filtered surface water were determined as 147 mg/L and 6.819 mg/L, respectively.
- Other: Water and associated sediment were combined at a ratio of ca. 160 g (wet weight): 1000 mL and stirred vigorously. The mixture was allowed to stand for approximately 5 minutes after which the water amended sediment layer above the settled solids was decanted, and TSS was measured. This initial mixture had an insufficient concentration of TSS for preparation of the test matrix, and the procedure was repeated, reducing the settling time to two minutes. The TSS concentration of the sediment-amended water layer was measured and adjusted to approximately 1 g /L by addition of the filtered water. A standard plate count proved microbial presence (2.0×10^4 cfu/mL)
Details on source and properties of sediment:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Water and associated sediments from the Brandywine Creek (Pennsylvania, USA). Sediment samples were taken from the entire 2 cm upper layer of the underwater sediment. The sediment was separated from the water by filtration and wet-sieved using a 2 mm-sieve.
- Storage conditions: Collection 19 September 2018, and were stored in a refrigerator at ca. 4 °C upon delivery to the testing
- Storage length: Less than 24 hours
- Textural classification (i.e. %sand/silt/clay): Not reported, however location on sediment collection provided in the full study report.
- pH at time of collection: Water pH 6.92
- Organic carbon (%): 10.14%
- Redox potential (mv) initial/final: Not reported
- CEC (meq/100 g): Not reported.
- Bulk density (g/cm³): Not reported.
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): See below for prepared combined water/sediment : A standard plate count proved microbial presence (2.0×104 cfu/mL)
- Sediment samples sieved: Yes (from water)
- Other: Water and associated sediment were combined at a ratio of ca. 160 g (wet weight): 1000 mL and stirred vigorously. The mixture was allowed to stand for approximately 5 minutes after which the water amended sediment layer above the settled solids was decanted, and TSS was measured. This initial mixture had an insufficient concentration of TSS for preparation of the test matrix, and the procedure was repeated, reducing the settling time to two minutes. The TSS concentration of the sediment-amended water layer was measured and adjusted to approximately 1 g /L by addition of the filtered water. A standard plate count proved microbial presence (2.0×10^4 cfu/mL)
Duration of test (contact time):
>= 0 - <= 58 d
Initial conc.:
0.5 µg/L
Based on:
act. ingr.
Remarks:
nominal [H-3]-test item ; biotic low and abiotic low concentrations
Initial conc.:
2.5 µg/L
Based on:
test mat.
Remarks:
nominal [H-3]-test item ; biotic high and abiotic high concentrations
Parameter followed for biodegradation estimation:
radiochem. meas.
other: Radiodistribution in Test Systems (e.g. mineralisation/loss due to volatilisation); kinetic parameters were also measured based on radiochem. measurements
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 250-mL serum glass bottles (calculated capacity: 280 mL) ; filled with 100 mL of water amended with sediment
- Composition of medium: See other fields.
- Additional substrate: Not applicable.
- Solubilising agent (type and concentration if used): Not applicable.
- Test temperature: At: 12 °C : 11.4 to 13.6 °C ; at 20 °C was 18.72 to 20.57 °C
- pH: 6.60 to 7.27 during the study
- pH adjusted: No.
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: No. Not required.
- Suspended solids concentration: The TSS concentration of the sediment-amended water layer was measured and adjusted to approximately 1 g/L
- Continuous darkness: Yes.
- Any indication of the test material adsorbing to the walls of the test apparatus: No. Overall mean mass balances were acceptable for all treatment groups, ranging from 95.5% of applied radioactivity (% AR) to 104.7% AR, with individual intervals ranging from 90.0% to 108.1% AR. Insignificant radioactivity recovery was observed in headspace traps regardless of the test.
systems.

TEST SYSTEM
- Culturing apparatus: 250-mL serum glass bottles (calculated capacity: 280 mL) ; filled with 100 mL of water amended with sediment
- Number of culture flasks/concentration: See below:
1. Abiotic sterile control: Sterilized Test Samples:
Low test concentration (ca. 0.5 μg/L) (12°C) (n=8)
High test concentration (ca. 2.5 μg/L) (20°C) (n=8)
Sterilized 3H-reference item High test concentration (ca. 2.5 μg/L) (20°C) (n=8)
2. Reference control:
Benzoic reference 3H benzoic acid (ca. 2.5 μg/ L) (20 °C) (n=14)
3. Test samples
Low test concentration (ca. 0.5 μg/L) (12°C) (n=24)
High test concentration (ca. 2.5 μg/L) (20°C) (n=24)
3H-reference item High test concentration (ca. 2.5 μg/L) (20°C) (n=24)
3H-reference item High test concentration (ca. 2.5 μg/L) (12°C) (n=8)
- Method used to create aerobic conditions: The pH and dissolved oxygen content in one sacrificial aerobic control chamber were measured on a weekly basis in Weeks 0 through 4, and in Weeks 6 through 8, in order to ensure the existence of aerobic conditions. The ranges of pH and dissolved oxygen content in control samples during the study were 6.60 to 7.27 and 8.45 to 9.07 mg/L, respectively. Aerobic conditions were maintained throughout the study.
- Method used to create anaerobic conditions: Not applicable.
- Method used to control oxygen conditions: See above.
- Measuring equipment:
1. HPLC fractionalisation
2. HPLC/β-RAM
3. Liquid Scintillation Counting (LSC), as applicable.
The radioactive distributions of parent test or reference substance and associated metabolites were determined by high performance liquid chromatography with a radio flow detector (HPLC-β-RAM). Mineralization of the radiolabeled test and reference substances (formation of tritiated water, 3H2O) was measured, when applicable, by HPLC fractionation and was presented as the volatile loss of the selected fraction at the retention time associated with 3H2O following drying.

- Test performed in closed vessels due to significant volatility of test substance: Yes. test vessels were sealed with silicone/PTFE septa and crimp caps.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used:
1. For the Headspace : water traps, triplicate aliquots were submitted for LSC analysis directly.
2. For the Headspace : PU trap, the PU plug was extracted by placing the plug inside the barrel of a disposable
syringe and adding 10 mL of acetonitrile. The acetonitrile was expelled from the PU trap by compressing it with the syringe plunger and triplicate aliquots were submitted for LSC analysis. Volatilized test or reference substance and their metabolites collected on a polyurethane foam plug and in a water trap from the headspace of test vessels presented ≤ 0.1% AR of the applied activity regardless of test systems and was considered negligible.
- Other:

SAMPLING
- Sampling frequency: Between six sampling intervals of three or 1 replicate (with 2 to 6 spares) were performed in test samples, sterilised test samples and reference items and/or five sampling intervals with 2 replicates plus four spares in the benzoic acid toxicity control. In the blank control weekly sampling intervals with 1 replicate scarified.
- Sampling method used per analysis type: At each sampling interval, individual test vessels from the test and reference dose groups were connected to the gas trapping system. The test samples were removed from the shaker table and connected to a gas trapping system consisting of a polyurethane (PU) foam plug to trap volatiles followed by water to trap 3H-water vapor. A syringe was used to inject 100 mL of acetonitrile into the test vessel through the septum cap. The syringe was then used to inject a 100-mL volume of air into the vessel to provide additional purging. The headspace gases were displaced through the gas trapping system.
After adding the acetonitrile and passing the displaced gases through the trapping system, each test vessel was disconnected from the trapping system, fitted with a new septum and crimp cap, and placed on a shaker table and shaken at 250 rpm for approximately 30 minutes. An aliquot of each sample solution was removed and centrifuged. Triplicate samples of the ACN-diluted test samples were analysed by LSC before and after centrifugation. The supernatants from the centrifuged samples were analysed by HPLC-β-RAM for radioactive distribution of the test or reference substance and metabolites.
- Sterility check if applicable: Yes.
- Sample storage before analysis: Analysed immediately
- Other: Further information above or below.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. Untreated (20°C) (n=10)
- Abiotic sterile control: Yes ; Sterilized Test Samples: Low test concentration (ca. 0.5 μg/L) (12°C) (n=8) and High test concentration (ca. 2.5 μg/L) (20°C) (n=8) and Sterilized 3H-reference item High test concentration (ca. 2.5 μg/L) (20°C) (n=8)
- Toxicity control: Yes. Benzoic reference 3H benzoic acid (ca. 2.5 μg/ L) (20 °C) (n=14)
- Other:
1. The aerobic controls were prepared and incubated analogous to the biotic test chambers, but were not dosed with test or reference substance. They were sealed with septa and crimp caps and incubated at 20 °C
2. Both Test samples Low test concentration (ca. 0.5 μg/L) (12°C) (n=24) and High test concentration (ca. 2.5 μg/L) (20°C) (n=24) and 3H-reference item High test concentration (ca. 2.5 μg/L) (20°C) (n=24) and 3H-reference item High test concentration (ca. 2.5 μg/L) (12°C) (n=8) were also performed in parallel to the controls, above.

STATISTICAL METHODS:
The following replicates (vessels) have been excluded from statistical calculations due to unacceptable (low) recoveries:
Day 2 / Test item abiotic / 2.5 μg/L vessel id: 58
Day 7 / Biotic, 0.5 μg/L vessel id: 9
Day 14 / Biotic, 0.5 μg/L vessel id: 12
Day 14 / Abiotic, 0.5 μg/L vessel id: 52
Day 21 / Biotic, 0.5 μg/L vessel: 14
The low recoveries were suspected to have been attributable to incorrect installation of the vessels' septa. These exclusions had no impact on results or integrity of the study due to sufficient replicates or data from other time intervals.

Statistical analysis included:
1. Mean Distribution of Parent and Transformation Products in Test Systems
2. Disappearance of Parent from the Biotic Test Systems (e.g. for DT50 and or rate constant determinations)
3. SFO Model Statistics for Disappearance of Parent from the Biotic Test Systems
4. Decline of Transformation Product in ROI 5 from the Biotic Test Systems
5. SFO Model Statistics for Decline of Transformation Product in ROI 5 from the Biotic Test Systems
6. Disappearance of Parent from the Abiotic Test Systems + related SFO Model Statistics (e.g. for DT50 and or rate constant determinations)
Equivalent statistics were also performed on all relevant systems (biotic, abiotic, test item, reference item etc).
All calculations were performed in Excel 2010 in full precision mode. Manual calculations were also performed.
Note: HPLC Peaks were grouped into regions of interest (ROI) based on typical retention times and chromatographic profiles. Regions of interest for the test and reference substances are described in the full study report.
Reference substance:
benzoic acid, sodium salt
Remarks:
Benzoic reference 3H benzoic acid (ca. 2.5 μg/ L) (20 °C) (n=14)
Reference substance:
other: The identity of the radiolabelled reference substance is given in the full study report
Remarks:
3H-reference item High test concentration (ca. 2.5 μg/L) (20°C) (n=24) and 3H-reference item High test concentration (ca. 2.5 μg/L) (12°C) (n=8)
Test performance:
- Overall mean mass balances were acceptable for all treatment groups, ranging from 95.5% of applied radioactivity (% AR) to 104.7% AR, with individual intervals ranging from 90.0% to 108.1% AR. Insignificant radioactivity recovery was observed in headspace traps regardless of the test.
- The recovery of applied radioactivity for each test system was within the acceptable range of 90-110%.
- Dose verification recoveries for the [3H]-test item treatment groups were 101.1% and 103.9% of nominal for the low and high dose levels, respectively. Recovery for the [3H]-reference item treatment groups was 102.6% of nominal. Recovery for the [3H]-Benzoic acid group was 106.8% of nominal. These were considered acceptable and % AR and mass balance calculations for treated vessels were not adjusted for verification recoveries.
- Insignificant radioactivity recovery was observed in headspace traps regardless of the test systems.
Key result
Compartment:
natural water / sediment: freshwater
DT50:
ca. 10.5 d
Temp.:
12 °C
Remarks on result:
other: the Simple First-Order (SFO) model provided acceptable fits to the data based on ² and r2 values, residual distribution, and visual assessment of the fitted curves
Remarks:
Biotic at 0.5 μg/L (12 °C)
Key result
Compartment:
natural water / sediment: freshwater
DT50:
ca. 10.8 h
Temp.:
12 °C
Remarks on result:
other: the Simple First-Order (SFO) model provided acceptable fits to the data based on ² and r2 values, residual distribution, and visual assessment of the fitted curves
Remarks:
Abiotic at 0.5 μg/L (12 °C)
Key result
Compartment:
natural water / sediment: freshwater
DT50:
ca. 21.1 d
Temp.:
20 °C
Remarks on result:
other: the Simple First-Order (SFO) model provided acceptable fits to the data based on ² and r2 values, residual distribution, and visual assessment of the fitted curves
Remarks:
Biotic at 2.5 μg/L (20 °C)
Key result
Compartment:
natural water / sediment: freshwater
DT50:
ca. 5.14 d
Temp.:
20 °C
Remarks on result:
other: the Simple First-Order (SFO) model provided acceptable fits to the data based on ² and r2 values, residual distribution, and visual assessment of the fitted curves
Remarks:
Abiotic at 2.5 μg/L (20 °C)
Transformation products:
not measured
Remarks:
The DT50 values represent rates for the initial steps of degradation of parent through presumed hydrolysis. The radio-labeling position on the molecule precludes assessment of any subsequent degradation pathway(s).
Details on transformation products:
The DT50 values represent rates for the initial steps of degradation of parent through presumed hydrolysis. The radio-labeling position on the (parent) molecule precludes assessment of any subsequent degradation pathway(s).
Evaporation of parent compound:
no
Remarks:
See below for further information
Volatile metabolites:
yes
Residues:
not specified
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): None that impacted the study integrity. See above sections for further details.

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: Not reported.
- Range of maximum concentrations in % of the applied amount at end of study period: Not reported.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: Not reported.
- Range of maximum concentrations in % of the applied amount at end of study period: Not reported.

The derived DT50 values represent rates for the initial steps of degradation of parent through presumed hydrolysis. The radio-labeling position on the molecule precludes assessment of any subsequent degradation pathway(s).

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: Overall mean mass balances were acceptable for all treatment groups, ranging from 95.5% of applied radioactivity (% AR) to 104.7% AR, with individual intervals ranging from 90.0% to 108.1% AR.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not applicable.
- % of applied amount at end of study period: Not applicable.

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not applicable.
- % of applied amount at end of study period: Not applicable.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Not known.
(i) The formation of 3H2O (mineralization) was determined by HPLC and was based on the relative quantity (% AR) of radioactivity at the retention time associated with 3H2O
(ii) Percent mineralization was calculated as the volatile loss × % AR of the 3H2O peak
The extent of mineralization, or tritiated water formation, as assessed by HPLC fractionation, was 79.8% and 63.1% for the low and high dose biotic test systems, respectively, by the end of the study.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: See above.
In the biotic systems, the major transformation product was observed in the retention time region associated with 3H2O, and was present at 83.1% AR and 65.7% AR, respectively, in the low and high dose groups by Day 58. One other peak was observed at >10% AR during the course of the study, and this occurred in the retention time region of ca, 4.5 to 5 minutes, prior to the parent substance, in both the low and high dose groups. By Day 58, this peak was not detectable in the low dose group, and had
declined to ca. 6% AR in the high dose group. No metabolite peaks other than that associated with the retention time of 3H2O were present at >10% by the end of the study.
In the abiotic systems, major metabolites (>10% AR) were detected in several polar regions prior to the parent test substance. These metabolites were attributed to an initial brief and transient abiotic process. No peaks were detected at the typical retention time of 3H2O, with the exception of one interval for the high dose system (<5% AR), indicating that no further degradation had occurred, and no or negligible mineralization had taken place in the abiotic systems, and that the degradation and subsequent mineralization observed in the biotic systems were primarily due to microbial activity.

STERILE TREATMENTS (if used)
- Transformation of the parent compound: Yes : abiotic and biotic transformation : transformation and subsequent mineralization, or tritiated water formation, of the test substance in the biotic water/sediment systems was attributed to microbial degradation, following an initial brief and transient abiotic component. Slightly slower degradation of test substance occurred in the biotic group at the higher treatment level of 2.5 μg/L.
- Formation of transformation products: Yes, although the rapid disappearance under abiotic conditions indicates that the first step is an abiotic process only. The appearance of a cluster of peaks in the polar regions of abiotic sample chromatograms, in combination with the lack of peaks in the region associated with 3H2O, indicate that no further degradation occurred in the abiotic systems, as expected.
- Formation of extractable and non-extractable residues: None reported.
- Volatilization:
For the biotic test systems: mean volatile losses fluctuated, but were generally consistent between sampling intervals, ranging from 52.9% to 64.4% for the 20 °C incubation group and 44.6% to 70.7% in the 12 °C incubation group.
In the abiotic test systems, losses were >94% percent at all intervals.
The lower volatile losses in the biotic groups were consistent with the rapid formation of polar metabolites, while the absence of any transformation in the abiotic systems indicated that the high volatile loss in those systems was caused by the reference substance volatilizing (known to have higher volatility than test item, per the study report).

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any): Not applicable.
Results with reference substance:
1. The extent of mineralization for biotic test systems dosed with [3H]-reference item at 2.5 μg/L and incubated at 20 °C or 12 °C was 34.8% AR and 49.7% AR, respectively.
2. Major metabolite peaks (>10% AR) were observed in the polar regions prior to the parent reference substance, including the region associated with 3H2O.
3. The difference in incubation temperatures (12 °C vs. 20 °C) had no effect on the degradation of the reference substance. The DT50 for the reference substance was <2 days at both temperatures.
4. The viability controls treated with radiolabeled benzoic acid demonstrated the presence of microbial activity (control). At a nominal concentration of 2.5 μg/L mineralization occurred at 95.4% AR by Day 2 and 97.9% AR by Day 14. Chromatographic analysis of the Day 14 samples confirmed total mineralization of the reference substance.

Table 1. Kinetic analyses of radio-labelled substance degradation in a water/sediment test system

Test System

Component

DT50 (days)

Rate constant (k)

[day-1]

Chi-squared

r2

Biotic at 0.5 µg/L

(12 °C)

Parent

10.5

0.06624

3.34

0.9976

ROI 5

10.8

0.06434

19

0.8984

 

 

 

 

 

Abiotic at 0.5 µg/L

(12 °C)

Parent

10.8

0.06435

9.81

0.9822

 

 

 

 

 

Biotic at 2.5 µg/L

(20 °C)

Parent

21.1

0.03293

4.87

0.9832

ROI 5

37.4

0.01856

4.29

0.8076

 

 

 

 

 

 

Abiotic at 2.5 µg/L

(20 °C)

Parent

5.14

0.1348

6.68

0.9968

ROI 4

49.3

0.01407

6.36

0.8947

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, transformation and subsequent mineralization of the test substance from the biotic water/sediment systems was attributed to microbial degradation following an initial brief and transient abiotic component. Slightly slower degradation of [3H]-test item occurred in the biotic group at the higher treatment level of 2.5 μg/L. DT50 values for parent were calculated as 10.5 and 10.8 days, respectively, for the low dose biotic and abiotic systems, and 21.1 and 5.14 days, respectively, for the high dose biotic and abiotic systems.
Executive summary:

The aerobic mineralisation in surface water : simulation biodegradation test was carried out according to OECD TG 309 guideline under GLP. The mineralization and degradation rates of the test substance : [3H]-test item were studied in biotic (live) and abiotic (sterile) water/sediment test systems. Radiolabelled test substance was applied to appropriate test system groups at nominal concentrations of 0.5 μg/L and 2.5 μg/L for the low and high dose groups, respectively. Radiolabelled reference substance, [3H]-reference item (the identity of which is given in the full study report), was applied to appropriate test systems at a nominal concentration of 2.5 μg/L. The low dose test substance vessels were incubated at approximately 12 °C, and high dose test substance vessels were incubated at approximately 20 °C. One biotic and one abiotic group of reference item vessels were incubated at approximately 20 °C, while a second biotic group was incubated at approximately 12 °C. Vessels were incubated for up to 58 days. The radioactive distributions of parent test or reference substance and associated metabolites were determined by high performance liquid chromatography with a radio flow detector (HPLC-β-RAM). Mineralization of the radiolabelled test and reference item substances (formation of tritiated water, 3H2O) was measured, when applicable, by HPLC fractionation and was presented as the volatile loss of the selected fraction at the retention time associated with 3H2O following drying. [3H]-Benzoic acid reference substance was also applied to appropriate test systems at a nominal concentration of 2.5 μg/L and the vessels were incubated at approximately 20 °C, to assess the activity and viability of the microbial population. Overall mean mass balances were acceptable for all treatment groups, ranging from 95.5% of applied radioactivity (% AR) to 104.7% AR, with individual intervals ranging from 90.0% to 108.1% AR. Insignificant radioactivity recovery was observed in headspace traps regardless of the test systems. Parent [3H]- test item disappeared steadily from all treated test systems during the course of the study. In the biotic systems, the major transformation product was observed in the retention time region associated with 3H2O, and was present at 83.1% AR and 65.7% AR, respectively, in the low and high dose groups by Day 58. One other peak was observed at >10% AR during the course of the study, and this occurred in the retention time region of ca. 4.5 to 5 minutes, prior to the parent substance, in both the low and high dose groups. By Day 58, this peak was not detectable in the low dose group and had declined to ca. 6% AR in the high dose group. No metabolite peaks other than that associated with the retention time of 3H2O were present at >10% by the end of the study. The extent of mineralization, or tritiated water formation, as assessed by HPLC fractionation, was 79.8% and 63.1% for the low and high dose biotic test systems, respectively, by the end of the study. In the abiotic systems, major metabolites (>10% AR) were detected in several polar regions prior to the parent test substance. These metabolites were attributed to an initial brief and transient abiotic process. No peaks were detected at the typical retention time of 3H2O, with the exception of one interval for the high dose system (<5% AR), indicating that no further degradation had occurred, and no or negligible mineralization had taken place in the abiotic systems, and that the degradation and subsequent mineralization observed in the biotic systems were primarily due to microbial activity. Parent [3H]-reference item disappeared rapidly from biotic systems and was not detected in samples from either system (20 °C or 12 °C incubation) on Days 2 through 58. Major metabolite peaks (>10% AR) were observed in the polar regions prior to the parent substance, including the region associated with 3H2O. The extent of mineralization, or tritiated water formation, as assessed by HPLC fractionation was 34.8% AR and 49.7% AR, respectively, in the 20 °C and 12 °C biotic reference item systems. In the abiotic [3H]-reference item system, no metabolites were observed at any interval during the course of study, indicating that the degradation and mineralization observed in the biotic test systems was due to microbial activity. Kinetic analyses were performed to estimate disappearance rates of [3H]-test item and selected metabolites (>10% AR and exhibiting a suitable pattern of decline for modelling) using CAKE software. For the reference systems dosed with 3H-benzoic acid at a nominal concentration of 2.5 μg/L, mineralization occurred at 95.4% AR by Day 2 and 97.9% AR by Day 14. Chromatographic analysis of the Day 14 samples confirmed total mineralization of the reference substance, verifying microbial viability. Transformation and subsequent mineralization of the test substance from the biotic water/sediment systems was attributed to microbial degradation following an initial brief and transient abiotic component. Slightly slower degradation of [3H]-test item occurred in the biotic group at the higher treatment level of 2.5 μg/L. DT50 values for parent were calculated as 10.5 and 10.8 days, respectively, for the low dose biotic and abiotic systems, and 21.1 and 5.14 days, respectively, for the high dose biotic and abiotic systems.

Description of key information

Biodegradation: rapidly (bio)degradable due to DT50 < 16 days in simulated environmentally relevant conditions; DT50 = 10.5 and 10.8 days, respectively for biotic and abiotic systems at 0.5 μg/L and 12 °C; slightly slower degradation of parent occurred in the biotic group at the higher treatment level of 2.5 μg/L with DT50 21.1 and 5.14 days respectively for biotic and abiotic systems at 2.5 μg/L and 20°C, OECD TG 309, 2020

Key value for chemical safety assessment

Half-life in freshwater:
10.5 d
at the temperature of:
12 °C
at the temperature of:
20 °C
Half-life in freshwater sediment:
10.5 d
at the temperature of:
12 °C
at the temperature of:
20 °C

Additional information

Key study : OECD TG 309, 2019 : The aerobic mineralisation in surface water : simulation biodegradation test was carried out according to OECD TG 309 guideline under GLP. The mineralization and degradation rates of the test substance : [3H]-test item were studied in biotic (live) and abiotic (sterile) water/sediment test systems. Radiolabelled test substance was applied to appropriate test system groups at nominal concentrations of 0.5 μg/L and 2.5 μg/L for the low and high dose groups, respectively. Radiolabelled reference substance, [3H]-reference item (the identity of which is given in the full study report), was applied to appropriate test systems at a nominal concentration of 2.5 μg/L. The low dose test substance vessels were incubated at approximately 12 °C, and high dose test substance vessels were incubated at approximately 20 °C. One biotic and one abiotic group of reference item vessels were incubated at approximately 20 °C, while a second biotic group was incubated at approximately 12 °C. Vessels were incubated for up to 58 days. The radioactive distributions of parent test or reference substance and associated metabolites were determined by high performance liquid chromatography with a radio flow detector (HPLC-β-RAM). Mineralization of the radiolabelled test and reference item substances (formation of tritiated water, 3H2O) was measured, when applicable, by HPLC fractionation and was presented as the volatile loss of the selected fraction at the retention time associated with 3H2O following drying. [3H]-Benzoic acid reference substance was also applied to appropriate test systems at a nominal concentration of 2.5 μg/L and the vessels were incubated at approximately 20 °C, to assess the activity and viability of the microbial population. Overall mean mass balances were acceptable for all treatment groups, ranging from 95.5% of applied radioactivity (% AR) to 104.7% AR, with individual intervals ranging from 90.0% to 108.1% AR. Insignificant radioactivity recovery was observed in headspace traps regardless of the test systems. Parent [3H]- test item disappeared steadily from all treated test systems during the course of the study. In the biotic systems, the major transformation product was observed in the retention time region associated with 3H2O, and was present at 83.1% AR and 65.7% AR, respectively, in the low and high dose groups by Day 58. One other peak was observed at >10% AR during the course of the study, and this occurred in the retention time region of ca. 4.5 to 5 minutes, prior to the parent substance, in both the low and high dose groups. By Day 58, this peak was not detectable in the low dose group and had declined to ca. 6% AR in the high dose group. No metabolite peaks other than that associated with the retention time of 3H2O were present at >10% by the end of the study. The extent of mineralization, or tritiated water formation, as assessed by HPLC fractionation, was 79.8% and 63.1% for the low and high dose biotic test systems, respectively, by the end of the study. In the abiotic systems, major metabolites (>10% AR) were detected in several polar regions prior to the parent test substance. These metabolites were attributed to an initial brief and transient abiotic process. No peaks were detected at the typical retention time of 3H2O, with the exception of one interval for the high dose system (<5% AR), indicating that no further degradation had occurred, and no or negligible mineralization had taken place in the abiotic systems, and that the degradation and subsequent mineralization observed in the biotic systems were primarily due to microbial activity. Parent [3H]-reference item disappeared rapidly from biotic systems and was not detected in samples from either system (20 °C or 12 °C incubation) on Days 2 through 58. Major metabolite peaks (>10% AR) were observed in the polar regions prior to the parent substance, including the region associated with 3H2O. The extent of mineralization, or tritiated water formation, as assessed by HPLC fractionation was 34.8% AR and 49.7% AR, respectively, in the 20 °C and 12 °C biotic reference item systems. In the abiotic [3H]-reference item system, no metabolites were observed at any interval during the course of study, indicating that the degradation and mineralization observed in the biotic test systems was due to microbial activity. Kinetic analyses were performed to estimate disappearance rates of [3H]-test item and selected metabolites (>10% AR and exhibiting a suitable pattern of decline for modelling) using CAKE software. For the reference systems dosed with 3H-benzoic acid at a nominal concentration of 2.5 μg/L, mineralization occurred at 95.4% AR by Day 2 and 97.9% AR by Day 14. Chromatographic analysis of the Day 14 samples confirmed total mineralization of the reference substance, verifying microbial viability. Transformation and subsequent mineralization of the test substance from the biotic water/sediment systems was attributed to microbial degradation following an initial brief and transient abiotic component. Slightly slower degradation of [3H]-test item occurred in the biotic group at the higher treatment level of 2.5 μg/L. DT50 values for parent were calculated as 10.5 and 10.8 days, respectively, for the low dose biotic and abiotic systems, and 21.1 and 5.14 days, respectively, for the high dose biotic and abiotic systems.

 

Under the conditions of this study, transformation and subsequent mineralization of the test substance from the biotic water/sediment systems was attributed to microbial degradation following an initial brief and transient abiotic component. Slightly slower degradation of [3H]-test item occurred in the biotic group at the higher treatment level of 2.5 μg/L. DT50 values for parent were calculated as 10.5 and 10.8 days, respectively, for the low dose biotic and abiotic systems, and 21.1 and 5.14 days, respectively, for the high dose biotic and abiotic systems.

 

References:

1. OECD TG 309 (2004)

2. ECHA Guidance R.7b, (v4.0, June 2017), Section 7.9.4

3. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017), Annex II: Rapid Degradation and section II.2.3.1 Aquatic simulation tests

4. ECHA Guidance R.11 : PBT/vPvB assessment (v3.0, June 2017), Section 11.4.1.1.3