1-[4-(1,1-dimethylethyl)phenyl]-3-(4-methoxyphenyl)propane-1,3-dione

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:

key study

Study period:

2019-07-16 to 2020-06-10

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:

April 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical name (IUPAC): 1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3-dione
- CAS number: 70356-09-1

Radiolabelling:

yes

Remarks:

[methoxyphenyl-ring-U-14C]BMDBM and [tert-butyl-phenyl-ring-U-14C]BMDBM

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Remarks:

Rhine river water/sediment system

Details on source and properties of surface water:

- Site: Rhein at Mumpf (4332 Mumpf, Switzerland)
- Coordinates: 47.546024° N, 7.931460 °E
- Source of inoculum: sediment and water
- Sampling date: July 16, 2019
- Storage conditions: water-logged (>5 cm water layer) at about 4 °C in the dark
- Storage length: for up to 20 days until use
- Temperature at site: 21.2 °C
- pH (water): 8.25
- Oxygen concentration: 8.98 mg/L
- Redox potential: 433 mV
- Turbidity: not available

Details on source and properties of sediment:

- Site: Rhein at Mumpf (4332 Mumpf, Switzerland)
- Coordinates: 47.546024° N, 7.931460 °E
- Source of inoculum: sediment and water
- Sampling date: July 16, 2019
- Storage conditions: water-logged (>5 cm water layer) at about 4 °C in the dark
- Storage length: for up to 20 days until use
- Depth (sediment layer): 5-10
- Colour: greyisch brown
- Smell: slightly moldy

Duration of test (contact time):

178 d

Initial conc.:

15 µg/L

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Test temperature: 10.0 +/- 0.14 °C

SAMPLING
- Sampling frequency: duplicate samples of the Rhine river water/sediment system were taken immediately after application (0 h) and after 0.5, 1, 2, 3, 7, 14, 28, 60, 91, 120, 154, and 178 days of incubation.

Test performance:

Parameters during acclimatization: Adequate stability of all systems, as reflected by pH, oxygen concentration in water and redox potential of sediment and water, was reached during the two weeks acclimatisation period. Redox potentials and oxygen concentrations measured in the water phase of treated samples were indicative for aerobic conditions during the incubation period. Redox potentials in the sediment were indicative for anaerobic conditions. Similar values were found in the untreated controls, demonstrating that the test item had no significant effects on the physico-chemical parameters of the test system pH values of the water and sediment phase indicated stable conditions in both systems during the whole incubation period. The purity of [14C]BMDBM-MP was determined by HPLC to be 99.4% and 97.8% before and after application, and for [14C]BMDBM-TP in the application solution it was purity and stability of the test item during the application procedure. Total mean recoveries were 94.7 ± 2.6% (MP-label) and 95.9 ± 2.9% AR (TP-label) for river under aerobic conditions.

Compartment:

natural water / sediment

% Recovery:

94.7

St. dev.:

2.6

Remarks on result:

other: label 1 - [methoxyphenyl-ring-U-14C]BMDBM (MP-label)

Compartment:

natural water / sediment

% Recovery:

95.9

St. dev.:

2.9

Remarks on result:

other: label 2 - [tert-butyl-phenyl-ring-U-14C]BMDBM (TP-label)

Key result

Compartment:

natural water: freshwater

DT50:

0.13 d

Type:

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

Temp.:

12 °C

Key result

Compartment:

natural sediment: freshwater

DT50:

111 d

Type:

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

Temp.:

12 °C

Key result

Compartment:

natural water / sediment: freshwater

DT50:

72.1 d

Type:

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

Temp.:

12 °C

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

Up to 8 and 10 degradation products were detected in the water phase of label 1 and 2, respectively.
M1 represented the major degradation product in the water phase of label 1 reaching a maximum of 3.0 % AR on DAT 28 and amounting to 0.4 % AR on DAT 178. Seven additional degradation products were detected only at minor amounts in the water phase of label 1 reaching a maximum of 0.9 % AR (M4) on DAT 91.
M2 was the most prominent degradation product detected in the water phase of label 2. M2 reached a maximum of 4.8 % AR in the water phase on DAT 14. It decreased to 1.1 % AR on DAT 178. In addition, M3 was detected once at 1.7 % AR (DAT 91) while the other degradation products did not exceed 1.3 % AR (M11).
In the sediment phase, BMDBM reached a maximum amount of 67.6 % AR / 72.7 % AR (label 1 / 2) on DAT 7 and decreased to 26.4 % / 34.5 % AR (label 1 / 2) on DAT 178. Besides the parent, up to 3 metabolites were detected in the sediment phase for both labels. M3 represented the main degradation product for both labels reaching a maximum of 13.1 % / 14.8 % AR (label 1 / 2) on DAT 91 and 11.1 % / 9.2 % AR (label 1 / 2) on DAT 178.
M1 reached a maximum of 4.1 % AR on DAT 91 for label 1 and M2 was detected at 5.1 % AR on DAT 91 for label 2. One additional minor degradation product M4 (label 1) and M11 (label 2) reached a maximum amount 1.1 % / 1.4 % AR (label 1 / 2) in the sediment phase.
In the total system, M3 which was mainly present in the sediment amounting to a maximum of 13.8 % / 16.5 % AR (label 1 / 2) on DAT 91. At the end of incubation, it was detected at 11.5 % / 9.2 % AR for label 1 / 2. M1 and M2 were label specific degradation products reaching a maximum of 5.5 % and 7.1 % AR in label 1 and 2, respectively. On DAT 178 the respective amounts of M1 and M2 were 1.6 % AR and 2.9 % AR.
M1 and M2 were confirmed by LC/MS to be 4-methoxy benzoic acid and 4-tert-butylbenzoic acid, respectively. Metabolite M3 was characterised by LC-MS/MS and a structure proposed corresponding to 1- (4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione.

Evaporation of parent compound:

no

Volatile metabolites:

yes

Residues:

yes

Details on results:

Immediately after application (0 h), 54.1 % / 66.9 % AR (label 1 / 2) of the radioactivity was detected in the river water phase while 44.5 % / 33.7 % AR (label 1 / 2) was detected in the sediment. Radioactivity in the water phase continuously decreased to 1.4 % / 1.8 % AR (label 1 / 2) after 178 days of incubation. The corresponding extractable radioactivity from the sediment reached 38.7 % / 45.6 % AR for label 1 /2 after 178 days. Non-extractable radioactivity amounted to 30.7 % / 27.8 % AR (label 1 / 2) on DAT 178. In addition, increasing amounts of carbon dioxide were detected from day 7 onwards amounting to 27.2 % (label 1) and 19.9 % (label 2) on DAT 178. None of the volatile radioactivity exceeded 0.1 % AR.

Half-life of BMDBM in the river water and sediment compartment is estimated based the on amount of BMDBM detected in the corresponding compartment for both labels. Calculation of the half-life of BMDBM in the sediment is estimated on data from sampling points starting with the maximum value for BMDBM from DAT 7 onwards to DAT 154. SFO kinetic model was used for all calculations. 

 

Compartment	DT50 (days)	DT90 (days)
River water phase	0.13	8.4
River sediment phase (incl DAT 154)	111	368
River water/sediment system	72.1	303

Validity criteria fulfilled:

yes

Conclusions:

The half-life, DT50, of the parent compound in freshwater sediment was determined to be 111 days at 12 °C in a river water/sediment system. The half-life in the water phase was 0.13 days at 12 °C and from the entire river water/sediment system the DT50 was determined to be 72.1 days at 12 °C.

Executive summary:

The biodegradation of the test item in freshwater sediment was tested in a river water/sediment system according to OECD 308. The test item was labelled at 2 label positions - [methoxyphenyl-ring-U-14C]BMDBM and [tert-butyl-phenyl-ring-U-14C]BMDBM. The radiolabelled BMDBM was applied onto the water phase of the equilibrated samples of the Rhine river water / sediment system (Switzerland) at a target concentration of 15 µg/L. The treated water / sediment system was incubated at 10.0 +/- 0.14 °C under aerobic conditions. Duplicate samples of the river water/sediment system were taken immediately after application (0 h) and after 0.5, 1, 2, 3, 7, 14, 28, 60, 120, 154 and 178 (river system) days of incubation. Total recovery of the applied radioactivity was the following 94.7 ± 2.6 % AR (Label 1), 95.9 ± 2.9 % AR (label 2). In the river system, immediately after application (0 h), 54.1 % / 66.9 % AR (label 1/2) of the radioactivity was detected in the river water phase while 44.5 % / 33.7 % AR (label 1/2) was detected in the sediment. Radioactivity in the water phase continuously decreased to 1.4 % / 1.8 % AR (label 1/2) after 178 days of incubation. The corresponding extractable radioactivity from the sediment reached 38.7 % / 45.6 % AR for label 1/2 after 178 days. Non-extractable radioactivity amounted to 30.7 % / 27.8 % AR (label 1/2) on DAT 178. In addition, increasing amounts of carbon dioxide were detected from day 7 onwards amounting to 27.2 % (label 1) and 19.9 % (label 2) on DAT 178. None of the volatile radioactivity exceeded 0.1 % AR. M1 (4-methoxy benzoic acid) was the major degradation products for label 1 in the water phase. M2 (4-tert-butylbenzoic acid) was the major degradation product for label 2 in the water phase. Additionally, one major metabolite M3 was formed. This metabolite was observed for both labels reaching maximum amounts of 13.1 % AR (label 1) and 14.8 % AR (label 2) in the sediment extracts on 91 DAT. Metabolite M3 was characterised by LC-MS/MS and a structure proposed corresponding to 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione. 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) were also detected in the sediment extracts at maximum amounts of 4.1 % AR and 5.1 % AR, respectively. Furthermore, two additional unknown metabolites, one per label were detected not exceeding 1.6 % AR. With regard to the total river system, [methoxyphenyl-14C] and [tert-butyl-phenyl-BMDBM decreased from initially 94.7 % and 95.2 % AR (mean, time 0) to 26.8 % and 34.8 % by the end of incubation, respectively. Metabolites M1 (4-methoxybenzoic acid) and M2 (para-tert-butylbenzoic acid) reached a maximum of 5.5 % and 7.1 % AR for label 1 and 2, respectively. The major metabolite 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reached up to 13.8 % (label 1) and 16.5 % AR (label 2) on 91 DAT in the water-sediment system and decreased to 11.5 % AR and 9.2 % AR, respectively, by the end of incubation. Up to six additional unknown metabolites were detected at minor amounts in the river system of either label, one of them reaching a maximum of 2.5 % AR, while none of the others exceeded 0.4 % AR at any time of the study. In the sediment phase of the river system, the major degradation product for both labels was M3. Small amount of M1 and M2 were observed for the respective label. In the pond sediment phase, for label 1 degradation products M1 and M3 were detected. And for label 2 the main degradation products M2 and M3 were detected. Half-life of BMDBM in the river water and sediment compartment is estimated based on the amount of BMDBM detected in the corresponding compartment for both labels. Calculation of the half-life of BMDBM in the sediment is estimated on data from sampling points starting with the maximum value for BMDBM from DAT 7 onwards to DAT 178. SFO kinetic model was used for all calculations. The half-life, DT50, of the parent compound in freshwater sediment was determined to be 111 days at 12 °C in a river water/sediment system. The half-life in the water phase was 0.13 days at 12 °C and from the entire river water/sediment system the DT50 was determined to be 72.1 days at 12 °C.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-04 to 2019-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

label 1 - 14C[methoxybenzoyl-CO] BMDBM, label 2 - 14C[tert-butylbenzoyl-CO] BMDBM

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection:
location: River Rhein by Mumpf, Switzerland, 47°546024N/07°931466E,
depth: on the surface, 0 - 10 cm,
contamination history: area not subject to effluent discharges and located far from human activity
- Storage conditions: at about 4 °C in the dark, until use
- Temperature (°C) at time of collection: 19
- pH at time of collection: 8.29
- Colour: slightly greenish
- Turbidity: 2.8 FNU
- Redox potential (mV) initial: 258.7
- Oxygen concentration (mg/L) initial: 9.77
- Dissolved organic carbon (%): 1.3 ppm
- Water filtered: yes
- Type and size of filter used, if any: 0.1 mm sieve

Duration of test (contact time):

60 d

Initial conc.:

0.006 mg/L

Based on:

other: dpm 14C[methoxybenzoyl-CO] BMDBM (radioactivity)

Remarks:

low dose

Initial conc.:

0.016 mg/L

Based on:

other: dpm 14C[methoxybenzoyl-CO] BMDBM (radioactivity)

Remarks:

high dose and high dose sterile

Initial conc.:

0.005 mg/L

Based on:

other: dpm 14C[tert-butylbenzoyl-CO] BMDBM (radioactivity)

Remarks:

low dose

Initial conc.:

0.015 mg/L

Based on:

other: dpm 14C[tert-butylbenzoyl-CO] BMDBM (radioactivity)

Remarks:

high dose and high dose sterile

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 100 mL natural water
- Sterile conditions: For high dose sterile samples, river water was sterilised by an autoclave at 121 °C for 30 minutes and subsequently treated as done with the high dose samples.
- Additional substrate: no
- Solubilising agent: methanol
- Test temperature: 10.8 ± 0.8 °C
- pH: 7.76 - 8.18
- pH adjusted: no
- Aeration of dilution water: yes, each flask was aerated with moistened air.
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: yes

TEST SYSTEM
- Culturing apparatus: open gas-flow-system with 350 mL Erlenmeyer flasks
- Number of culture flasks/concentration: per label 20 flasks for the low dose and the high dose each, 10 flasks for the high dose sterile, 2 flasks for the reference substance, solvent control and the blank each
- Method used to create aerobic conditions: Aeration and agitation facilitated oxygen transfer from headspace to liquid, in a way that aerobic conditions were maintained.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Samples were connected to a trapping system equipped with a total of two absorption traps, one containing ethylene glycol and the other 2N NaOH (in this sequence) to trap organic volatiles and 14CO2, respectively.

SAMPLING
- Test item: Duplicate samples treated with the test item were taken from each test dose system (High Dose (HD) and Low Dose (LD)) and single samples from High Dose Sterile (HDS) test dose system immediately after treatment (time 0) and after 3, 7, 15, 32, 41 (label 1) / 42 (label 2) and 60 days of incubation. Per sampling interval the entire sample of 100 mL was subject to sample work-up.
- Reference substace: Aliquots of the aqueous samples treated with benzoic acid were removed from the flasks and analysed by LSC and HPLC immediately after treatment (time 0) and after 7 and 14 days.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Number of culture flasks/concentration: 2 flasks for the solvent control and the blank each

STATISTICAL METHODS: The degradation rate of the test item in surface water was calculated assuming a single first order (SFO) kinetics model using CAKE kinetics software (version 3.2). Chi-square and r2 values were calculated directly by the software.

Reference substance:

other: Benzoic acid [14C(U)]

Compartment:

entire system

% Recovery:

92

St. dev.:

6.6

Remarks on result:

other: high dose for methoxybenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Compartment:

entire system

% Recovery:

90.4

St. dev.:

6.7

Remarks on result:

other: high dose sterile for methoxybenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Compartment:

entire system

% Recovery:

89.8

St. dev.:

9.7

Remarks on result:

other: low dose for methoxybenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Compartment:

entire system

% Recovery:

91.4

St. dev.:

5.5

Remarks on result:

other: high dose for tert-butylbenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Compartment:

entire system

% Recovery:

89.1

St. dev.:

6.4

Remarks on result:

other: high dose sterile for tert-butylbenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Compartment:

entire system

% Recovery:

86.9

St. dev.:

5.3

Remarks on result:

other: low dose for tert-butylbenzoyl-labelled test item

Remarks:

aqueous phase + organic rinsings

Key result

% Degr.:

88.3

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 1, high dose, organic phase after partitioning

Key result

% Degr.:

47.2

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 1, high dose sterile, organic phase after partitioning

Key result

% Degr.:

94.7

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 1, low dose, organic phase after partitioning

Key result

% Degr.:

95.1

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 2, high dose, organic phase after partitioning

Key result

% Degr.:

46.8

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 2, high dose sterile, organic phase after partitioning

Key result

% Degr.:

97.8

Parameter:

radiochem. meas.

Sampling time:

60 d

Remarks on result:

other: label 2, low dose, organic phase after partitioning

Key result

Compartment:

natural water: freshwater

DT50:

10.8 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: high dose

Key result

Compartment:

natural water: freshwater

DT50:

10.2 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: low dose

Key result

Compartment:

natural water: freshwater

DT50:

54.7 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: high dose sterile

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#10

No.:

#10

No.:

#4

No.:

#6

Details on transformation products:

Please refer to the attached tables for the detailes representation of the formation and decline of each major and minor metabolite for each label and test dose.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
Mean oxygen concentrations in the aqueous phase: label 1: 8.80 mg/L in HD, 8.63 mg/L HDS, 8.79 mg/L in LD; label 2: 8.37 mg/L in HD, 8.43 mg/L in HDS, 8.41 mg/L in LD
- Anomalies or problems encountered: none

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Metabolite M4 for label 1, first obderved on Day 15 at 1.2 %, maximum observed % was on Day 60 - 18.4 %.
Metabolite M10 for label 1 first observed on Day 7 at 0.5 %, maximum % observed on Day 15 - 7.8 %.
Metabolite M6 for label 2, first obderved on Day 7 at 17.4 %, maximum observed % was on Day 60 - 49.8 %.
Metabolite M10 for label 2 first observed on Day 3 at 3.5 %, maximum % observed on Day 15 - 15.8 %.

- Range of maximum concentrations in % of the applied amount at end of study period:
Metabolite M4 for label 1 - 18.4 % at Day 60
Metabolite M10 for label 1 - 3.0 % at Day 60
Metabolite M6 for label 2 - 49.8 % at Day 60
Metabolite M10 for label 2 - 2.9 % at Day 60

MINOR TRANSFORMATION PRODUCTS
For detailed information on the minor transformation products please refer to the attached tables and Section 'Any other information on results incl. tables'

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 1.6 - 5.8 % depending on the dose and the label position. Under sterile conditions the formation of CO2 was minimal.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: up to 3.2 % AR was observed at the end of the test present as volatile orgnaics. Again unter sterile conditions minor % AR of volatiles was observed.

Results with reference substance:

The total recovery of the radioactivity for the reference item after 14 days was 85.9 ± 13.8 %.
The benzoic acid % AR at time 0 was 101.7 %. After 7 and 14 days no % AR could be detected anymore. Therefore, the reference item was completely degraded in 14 days and thus, the microbial activity of the test system was conformed.

Radiochemical purity and stability of the test item

The radiochemical purity of [¹⁴C] BMDBM (methoxybenzoyl label) was determined by HPLC to be 100.0 % HD (100 % LD) before and 100 % HD (100 % LD). Corresponding values for the tert-butylbenzoyl label were 100 % HD (100 % LD) and 100 % HD (100 % LD).

The HPLC results demonstrated stability of BMDBM (both labels) during the application procedure.

BMDBM appeared to form an equilibrium of two tautomers in solution designated as Parent-1 and -2; reference substance (R0-1 and R0-2).

 

Preliminary test

Based on the preliminary findings observed with the natural water tests, the sampling intervals in the definitive test were defined as follows, i.e. after 0, 3, 7, 15, 32, 41 and 60 days of incubation for ¹⁴C[methoxybenzoyl-CO] BMDBM (label 1) and after 0, 3, 7, 15, 32, 42 and 60 days of incubation for ¹⁴C[tert-butylbenzoyl-CO] BMDBM (label 2).

 

Microbialactivityof test water

Within 14 days of incubation, the mean amount of radioactivity applied as benzoic acid to the control samples decreased rapidly from the aqueous phase from initially 101.7 % to 19.4 % under formation of 65.4 % of radioactive carbon dioxide. Benzoic acid, with an initial mean amount of 101.7 % AR at time zero, degraded completely in the respective samples within 7 days of incubation.

Within 14 days of incubation, the mean amount of benzoic acid decreased rapidly in the aqueous phase from initially 101.3 % to 15.1 % under formation of 74.5 % of radioactive carbon dioxide. Benzoic acid, with an initial mean amount of 101.3 % AR at time zero, completely dissipated from the respective samples within 7 days of incubation.

As more than 90 % of benzoic acid degraded within 7 days in the solvent control samples, and evolved carbon dioxide was comparably observed during the 14-day incubation period in each of the test systems, the test water was considered to be microbially active.

 

Overall recovery of radioactivity

Methoxybenzoyl label

For the methoxybenzoyl-labelled test item, total mean recoveries were 92.0 ± 6.6 % AR (applied radioactivity) for the high dose, 90.4 ± 6.7 % AR for the high dose sterile and 89.8 ± 9.7 % AR for the low dose experiment. The total recovery of radioactivity decreased below 90 % AR from 32 DAT onwards. With progress of the study, additional rinsing steps with methanol were necessary to recover radioactivity adsorbed to the glass equipment. In addition increasing amount of radioactivity was detected in combustions of particles in the filters. The low recovery is assumed to be associated to the strong adsorption potential of the test item, which might have resulted in a strong and partially irreversible adsorption to glass walls and/or suspended solids as the study progressed. In addition, it is also assumed that a partial loss of volatile radioactivity may have occurred since an increasing amount of carbon dioxide was observed from 32 DAT onwards which could be associated with the mineralization of 4-methoxy-benzoic acid being detected as one of the major metabolites of BMDBM. A low recovery of applied radioactivity has often been experienced in experiments investigating the degradation of benzoic acid or similar compounds and is even confirmed in this study with benzoic acid itself being used to test the microbial activity of the test water.

Immediately after treatment (time 0), mean values of 96.5 %, 103.2 % and 94.7 % AR (mean amounts) were measured in the high dose, high dose sterile and low dose samples, respectively (total of aqueous and organic phase after partitioning). Due to the strong hydrophobic nature of the test item, the majority of the applied radioactivity was extracted into the organic phase by partitioning. As the study progressed, increasing amounts of radioactivity, especially in the non-sterile samples, remained in the aqueous phase after the partitioning step/liquid-liquid extraction. After 60 days of incubation, the mean amount of radioactivity in the organic phase (including the acetonitrile rinse) of the respective samples represented 40.1 %, 61.0 % and 31.8 % AR, whereas 19.8 %, 3.2 % and 20.3 % remained in the aqueous phase of the corresponding samples. Additionally, 17.3 %, 22.5 % and 16.7 % AR were recovered from the high dose, high dose sterile and low dose samples by vessel/filter rinsings with methanol on DAT 60, respectively. As the study progressed, radioactivity bound to suspended solids increased. Therefore, filters were combusted from day 32 onwards. Amounts of 5.7 %, 4.0 % and 12.6 % AR were detected in combusted filters of HD, HD sterile and LD samples, respectively, at the end of the study (60 DAT).

Formation of radioactive carbon dioxide (¹⁴CO₂) increased throughout incubation in the high dose and low dose test systems, representing maximum mean amounts of 14.7 % and 10.6 % AR, respectively on 41 DAT. However, on DAT 60, the amount of radioactive carbondioxide dropped to 5.2 % and 5.2 % AR in high dose and low dose samples, respectively. In the respective sterile system, only negligible amounts of 0.1 % AR ¹⁴CO₂ were observed. Volatile products other than¹⁴CO₂ did not exceed 0.3 % AR in any methoxybenzoyl-labelled sample throughout the study.

Tert-butylbenzoyl label

For the tert-butylbenzoyl-labelled test item, total mean recoveries were 91.4 ± 5.5 % AR for the high dose, 89.1 ± 6.4 % AR for the high dose sterile and 86.9 ± 5.3 % AR for the low dose experiment. Similarly, as observed for the methoxybenzoyl label, overall recoveries were rather low also for the tert-butylbenzoyl label. Throughout the study an increasing amount of radioactivity was recovered in methanol rinsings of glass equipment and filters and by combustion of the filters. For this reason the low recovery of the applied radioactivity is assumed to be caused by the strong adsorption potential of the test item, which might have resulted in a partially irreversible adsorption to glass walls and/or suspended solids in the filters as the study progressed.

In addition, a loss of volatile radioactivity, most probably radioactive carbon dioxide is assumed to have occurred from mineralization of para-tert-butylbenzoic acid as one of the major metabolites since a slight increase of radioactive carbon dioxide was observed in parallel with the decrease of the recovered radioactivity during the progress of the study.

Low recovery of applied radioactivity can be often observed in experiments investigating the degradation of benzoic acid or similar compounds and is even confirmed in this study with benzoic acid itself being used to demonstrate the microbial activity of the test water.

Immediately after treatment (time 0), mean values of 93.2 %, 93.1 % and 93.6 % AR were measured in the high dose, high dose sterile and low dose samples, respectively (total of aqueous and organic phase after partitioning). Similarly, as observed with the methoxybenzoyl label, almost the entire applied radioactivity was initially extracted into the organic phase by partitioning. As the study progressed, increasing amounts of radioactivity, especially in the non-sterile samples, remained in the aqueous phase after the partitioning steps. On day 60, the mean amount of radioactivity in the organic phase (including the acetonitrile rinse) represented 60.6 %, 72.0 % and 56.1 % AR for high dose, high dose sterile and low dose samples, respectively, whereas 5.4 %, 0.9 % and 6.3 % remained in the aqueous phase of corresponding samples. Additional mean amounts of 4.0 %, 14.8 % and 9.1 % AR were recovered from the respective HD, HD sterile and LD samples by vessel rinses with methanol on day 60. Combustion of the filters (performed from day 32 onwards) recovered up to 13.6 %, 4.3 % and 10.1 % AR in HD, HD sterile and LD samples on day 60, respectively.

Formation of radioactive carbon dioxide (¹⁴CO₂) increased only slightly throughout incubation in the high dose and low dose test systems, representing maximum mean amounts of 6.5 % and 7.7 % AR, respectively on 42 DAT but dropped again to 2.1 % and 1.6 % AR, respectively on DAT 60.

 

Degradation of BMDBM and pattern of metabolite formation

BMDBM degraded faster in non-sterile samples than in the sterile natural water samples. Degradation observed in non-sterile samples resulted also in formation of higher amounts of radioactive carbon dioxide and higher number of metabolites when compared with sterile samples.

 

Methoxybenzoyl label

The mean amount of methoxybenzoyl-labelled BMDBM extracted into the organic phase including the acetonitrile rinse of the high dose system, decreased from initially 96.0 % AR at time zero to 11.7 % AR on 60 DAT. In the sterile high dose system, corresponding values were 102.7 % and 52.8 % AR. In the low dose system, the test item in the organic phase decreased to 5.3 % AR after 60 days of incubation.

Aside from the test item, in total up to thirteen metabolites were detected in high dose and nine in the low dose samples, whereas only up to seven metabolites were detected in the sterile system.

The most predominant metabolite in both non-sterile systems was M4 amounting to 8.8 % and 5.8 % AR in high dose and low dose samples on 15 DAT, respectively. M4 reached maximum amounts up to 18.4 % and 22.6 % AR on day 60 in high and low dose systems, respectively. In the sterile samples M4 was present at lower amounts from DAT 15 onwards reaching a maximum of 6.7 % on DAT 41. Another significant metabolite was M10, which was detected at a maximum of 7.8 % AR on day 15 in the high dose and at up to 10.7 % AR (32 DAT) in the low dose samples. On day 60, M10 was present at 3.0 % and 3.9 % AR in the high dose and low dose samples, respectively. Another radioactive fraction, M2, was detected at 11. 8% on day 15 in the high dose samples only. As this fraction was observed only once in significant amounts under any conditions, it was rather considered as a minor intermediate metabolite. For this reason, it was not characterized any further. All other metabolites formed were not regarded as relevant as they were only present at low amounts not exceeding once a maximum mean amount of 4.1 % (M1) AR. In sterile high dose samples a similar metabolite pattern as for non-sterile samples was observed with M4 and M10 appearing to be the main metabolites.

Metabolites M4 and M10 were characterized by LC-MS/MS. M4 was identified as 4-methoxy-benzoic acid and confirmed by the corresponding reference compound. Based on the mass fragments, M10 was suggested to be likely a reduced form of BMDBM namely 3-(4-tert-butyl-phenyl)-3-hydroxy-1-(4-methoxy-phenyl)-propan-1-one or 1-(4-tert-butyl-phenyl)-3-hydroxy-3-(4-methoxy-phenyl)-propan-1-one.

TLC analysis of the combined methanol rinses revealed that the radioactivity recovered in these steps represented mainly BMDBM and M4. As this radioactivity was recovered from different kind of surfaces, it was considered as not available to the system. Furthermore, the radioactivity present in the individual rinses was very low. Therefore, it was not taken into account for any pattern tables nor degradation kinetics.

The radioactivity remaining in the aqueous phase after the partitioning steps was characterized by TLC for the interval 60 DAT. The results showed that the aqueous phase consisted of six radioactive fractions, one of them representing M4. Parent was only present at a minor amount of about 0.25 % AR. The main radioactive fraction (TLC fraction 3) was scraped off the plate, the radioactivity eluted with methanol, and the resulting extract analysed by stop flow HPLC. The unknown fraction splitted up in two peaks representing 3.7 % and 9.0 % AR.

In addition, an attempt was made to characterize the radioactivity remaining in the aqueous phase for interval 60 DAT after liquid/liquid extraction by using LC-MS/MS. The MS/MS results confirmed that one of the radioactive fractions present was M4, which did not entirely partition in the organic phase. However, it was not possible to successfully characterize the other fractions in the aqueous phase, mainly due to severe interference of the highly concentrated matrix with the LC-MS causing an accumulative increase of backpressure of the system. The LC-MS analysis could not be repeated as the limited amount of sample was used up by several analysis. It is therefore concluded that the radioactivity remaining in the aqueous phase of the methoxybenzoyl label treated samples consisted of several unknown components, with the individual components representing up to 9.0 % AR (one single component).

 

Tert-butylbenzoyl label

The mean amount of tert-butylbenzoyl-labelled BMDBM extracted into the organic phase including the acetonitrile rinse of the high dose system, decreased from initially 92.6 % AR at time zero to 4.9 % AR on 60 DAT. In the sterile high dose system, corresponding values for the test item were 92.8 % and 53.2 % AR. In the low dose system, the test item in the organic phase decreased from 92.5 % AR at time 0 to 2.2 % AR after 60 days of incubation.

Aside from the test item, in total up to seven metabolites were detected in the different systems. The most predominant metabolite present in sterile and non-sterile systems was M6 representing amounts of 17.4 %, 10.7 % and 22.0 % AR in high dose, sterile and low dose samples on 15 DAT, respectively. M6 reached maximum amounts up to 49.8 %, 11.5 % and 50.5 % AR in high, high dose sterile and low dose systems on 60 DAT, respectively. The other significant metabolite was M10, which was detected up to 15.8 %, 5.2 % and 11.5 % AR in the high dose, high dose sterile and low dose samples on 15 DAT, respectively. The amount of M10 decreased throughout the study to 2.9 % and 3.3 % AR in the high dose and low dose samples on 60 DAT, respectively. One other unknown metabolite M2 was only detected once reaching an amount of 7.9 % and 7.8 % AR on 15 DAT in high dose and low dose samples, respectively. All other metabolites formed were not exceeding mean amounts of 4.2 % AR. In sterile samples unknown metabolites M1 and M2 were detected at maximum amounts of 10.8 % and 7.4 % AR on 15 DAT, respectively. No other major metabolites were detected in the sterile samples.

Metabolite M6 was identified to be para-tert-butylbenzoic acid by LC-MS/MS. In addition it was confirmed by LC-MS and TLC by co-elution with the commercial reference substance para-tert-butylbenzoic acid.

As for label 1, M10 was also confirmed by LC-MS/MS in high dose samples of label 2. Based on the mass fragments, the molecular structure was suggested to be 3-(4-tert-butyl-phenyl)-3-hydroxy-1-(4-methoxy-phenyl)-propan-1-one or 1-(4-tert-butyl-phenyl)-3-hydroxy-3-(4-methoxy-phenyl)-propan-1-one. 

Radioactivity remaining in the aqueous phase after the partitioning step represented about 5 % AR in the high dose samples on DAT 60. This radioactivity was analysed by TLC revealing that no parent but six radioactive fractions were present with one dominant fraction amounting to about 2.9 % AR. Therefore, the radioactivity was not characterized any further.

 

Rate of degradation/dissipation of BMDBM

The degradation/dissipation rate for BMDBM in river water was calculated using a single first-order (SFO) kinetics model. The calculated degradation half-lives (DT₅₀) and DT₉₀ (normalized to 12 °C) values obtained for the total system are shown in the table below.

 

Test conc.	Model	M0	Parameter	Prob > t^a)	DT50 (days)	DT90 (days)	r^2	χ^2error %
High dose	SFO Parent	97.93	k = 0.05851	3.31E-014	10.8	35.8	0.9652	8.74
High dose sterile	SFO Parent	83.1	k = 0.01151	3.35E-004	54.7	181.7	0.6574	11.3
Low dose	SFO Parent	96.4	k = 0.06184	7.91E-011	10.2	33.8	0.9372	6.75

a) In order to assess the fitted degradation rates as statistically acceptable Prob> t (i.e. the p-value) should be < 0.05.

Note:  SFO kinetics was applied using CAKE software (version 3.2). DT₅₀: Calculated degradation half-life of parent. χ²: chi-square statistical value.

 

In conclusion, BMDBM was degraded to three major metabolites M4, M6 and M10. Besides several minor unknown metabolites also minor amounts of carbon dioxide were detected. Degradation in non-sterile water systems occurred to be faster than in sterilized natural water indicating a microbially mediated transformation. The half-life (DT₅₀) of BMDBM in biologically active surface water system was 10.8 days for the high dose (0.015 mg/L) and 10.2 days for the low dose (0.005 mg/L) test systems. The half-life in sterile systems was 54.7 days.

Due to the hydrophobic properties of BMDBM an increasing amount of radioactivity was found to be adsorbed to surfaces and suspended particles with progress of the study. Adsorbed radioactivity was found to consist of parent and metabolite M4 or M6. 

Two of the three major metabolites, 4-methoxy-benzoic acid (M4) and para-tert-butylbenzoic acid (M6) were confirmed by LC-MS/MS using commercial reference substances. Based on LC-MS/MS analysis, for M10, a suggestion was made for 3-(4-tert-butyl-phenyl)-3-hydroxy-1-(4-methoxy-phenyl)-propan-1-one. 4-methoxy-benzoic acid and para-tert-butylbenzoic acid were label specific and were formed by cleavage of the bond between the two aromatic rings while 3-(4-tert-butyl-phenyl)-3-hydroxy-1-(4-methoxy-phenyl)-propan-1-one represents a reduced form of BMDBM.

In general total recovery of radioactivity appeared to be lower than 90 % for some samples from DAT 32 onwards. Low recoveries were interpreted to be caused by a loss of radioactivity due to strong sorption of the parent to surfaces and particles as well as by partial loss of radioactive carbon dioxide from mineralization of the major metabolites. 

 

Test conc.	Model	M0	Parameter	Prob > t^a)	DT50 ^b) (days)	DT90 ^b) (days)	r^2	x^2 error %
M 10 high dose	SFO	11.59	k = 0.03895	2.16E-004	16.2	53.7	0.6733	7.66

a) In order to assess the fitted degradation rates as statistically acceptable Prob> t (i.e. the p-value) should be < 0.05.
b) Normalised to 12 °C, experimental data were obtained at 10.8 °C

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the half-life (DT50) of BMDBM normalized to 12 °C in biologically active surface water system was 10.8 days for the high dose (0.015 mg/L) and 10.2 days for the low dose (0.005 mg/L) test systems. Hence, degradation in biological active systems is not considered to be concentration dependent. In sterile samples, degradation occurred at a slower rate showing a half-life of 54.7 days. The faster degradation in non-sterile conditions may result from a combination of biotic and abiotic degradation of BMDBM. However based on the fact that degradation in the sterile system appeared to be much slower, it is concluded that the degradation was predominately a biotic degradation. In order to estimate the degradation rate for the biotic degradation per se, the degradation constant obtained for sterile samples was subtracted from the degradation rate obtained for non-sterile samples resulting in a DT50 value of 13.4 days and DT90 values of 44.5 days for biotic degradation at 12 °C. Due to the hydrophobic properties of BMDBM, an increasing amount of radioactivity was found to be adsorbed to surfaces and suspended particles with progress of the study. Due to the slower degradation of BMDBM in sterile systems, adsorption could play a more pronounced role as the parent compound shows a higher adsorption potential than the more polar metabolites. Therefore, the values calculated for the biotic degradation process can be considered as an estimation only.

Executive summary:

Aerobic mineralisation of [¹⁴C] BMDBM in surface water was investigated under defined laboratory conditions in the dark in accordance with OECD 309. For this purpose the radiolabelled test item, separately labelled in two positions (methoxybenzoyl and tert-butylbenzoyl position), was applied to 100 mL of natural river water at nominal test item concentrations of 0.016 and 0.006 mg/L for methoxybenzoyl label and 0.015 and 0.005 mg/L for tert-butylbenzoyl-labelled test item. Additionally, the high concentration experiment conducted with the both labels was performed under sterile conditions in order to gain information about abiotic degradability of the test item. The test flasks were incubated for a period of 60 days at 10.8 ± 0.8 °C under aerobic conditions by gently stirring the water. Radiolabelled benzoic acid (with and without solvent control) was used as reference substance to check for sufficient microbial activity of the test water.

The freshly collected water samples from the river Rhein were pretreated and prepared for the main experiment. Based on the findings of a preliminary test, the sampling intervals in the definitive test were defined as follows, i.e. after 0, 3, 7, 15, 32, 41 and 60 days of incubation for ¹⁴C[methoxybenzoyl-CO] BMDBM and after 0, 3, 7, 15, 32, 42 and 60 days of incubation for ¹⁴C[tert-butylbenzoyl-CO] BMDBM.

As a result of the high log Kow of the test item, the compound was found to be adsorbed to any surfaces and suspended particles throughout the incubation time. Despite of its relatively high specific radioactivity, only limited amounts of dpm could be applied to the samples, especially for the low dose samples. In order to minimize loss due to test item adsorption,the samples were treated with a changing work-up to recover as much as possible of the applied radioactivity (AR). Several analytical methods, like HPLC, LC-MS/MS, radio-HPTLC, LSC, were used for the determination of the test item concentrations and % AR.

For the methoxybenzoyl-labelled test item, total mean recoveries were 92.0 ± 6.6 % AR for the high dose, 90.4 ± 6.7 % AR for the high dose sterile and 89.8 ± 9.7 % AR for the low dose experiment. The total recovery of radioactivity decreased below 90 % AR from 32 DAT onwards. The low recovery is assumed to be associated to the strong adsorption potential of the test item, which might have resulted in a strong and partially irreversible adsorption to glass walls and/or suspended solids as the study progressed. In addition, it is also assumed that a partial loss of volatile radioactivity may have occurred from mineralisation of 4-methoxy-benzoic acid (M4) as one of the major metabolites since an increasing amount of carbon dioxide was observed from 32 DAT onwards. Formation of radioactive carbon dioxide (¹⁴CO₂) increased throughout incubation in the high dose and low dose test systems, representing maximum mean amounts of 14.7 % and 10.6 % AR, respectively on 41 DAT. In the respective sterile system, only negligible amounts of 0.1 % AR ¹⁴CO₂ were observed. Volatile products other than ¹⁴CO₂ did not exceed 0.3 % AR in any methoxybenzoyl-labelled sample throughout the study.

Aside from the test item, in total up to thirteen metabolites were detected in the organic phase of the high dose and nine in the low dose samples, whereas only up to seven metabolites were detected in the sterile system.The most predominant metabolite in both non-sterile systems was M4 which reached maximum amounts up to 18.4 % and 22.6 % AR on day 60 in high and low dose systems, respectively. Metabolite M4 was identified by LC-MS/MS to be 4-methoxy-benzoic acid using the commercial reference item.

For the tert-butylbenzoyl-labelled test item, total mean recoveries were 91.4 ± 5.5 % AR for the high dose, 89.1 ± 6.4 % AR for the high dose sterile and 86.9 ± 5.3 % AR for the low dose experiment. Again adsorption to the glass was observed and it is also assumed that a minor loss of volatile radioactivity may have occurred eg by mineralization of of 4-tert-butylbenzoic acid (M6) occurring as as one of the major metabolites. Formation of radioactive carbon dioxide (¹⁴CO₂) increased up to 6.5 % AR in the high and 7.7 % in the low dose systems on 42 DAT. In the respective sterile system, no significant formation of ¹⁴CO₂ was observed (maximum mean amounts of 0.3 % AR). Volatile products other than ¹⁴CO₂ did not exceed 3.2 % AR in any tert-butylbenzoyl-labelled sample throughout the study.

Aside from the test item, in total up to seven metabolites were detected in the different systems.The most predominant metabolite present in sterile and non-sterile systems was M6 which reached maximum amounts up to 49.8 %, 11.9 % and 50.5 % AR in high, high dose sterile and low dose systems on DAT 60, respectively. Metabolite M6 was identified by LC-MS/MS as para-tert-butylbenzoic acid by using the commercial reference item.

Radioactivity remaining in the aqueous phase after the partitioning steps represented about 5 % AR on interval 60 DAT. This radioactivity was analysed by TLC for one sample revealing that six minor components were remaining in the aqueous phase. Therefore, no further characterization was deemed necessary.

In conclusion, BMDBM occurred to degrade faster in non-sterile water systems than in sterilized natural water system indicating a microbially mediated degradation process. The half-life (DT₅₀) in biologically active surface water system was 10.8 days for the high dose (0.015 mg/L) and 10.2 days for the low dose (0.005 mg/L) test systems. The half-life in sterile systems was 54.7 days.

Reference 3

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-16 to 2020-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

[methoxyphenyl-ring-U-14C]BMDBM (MP-label) and [tert-butyl-phenyl-ring-U-14C]BMDBM (TP-label)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Remarks:

Pond water/sediment system

Details on source and properties of surface water:

- Site: Rhein at Mumpf (4332 Mumpf, Switzerland)
- Coordinates: 47.546024° N, 7.931460 °E
- Source of inoculum: sediment and water
- Sampling date: July 16, 2019
- Storage conditions: water-logged (>5 cm water layer) at about 4 °C in the dark
- Storage length: for up to 20 days until use
- Temperature at site: 21.2 °C
- pH (water): 8.25
- Oxygen concentration: 8.98 mg/L
- Redox potential: 433 mV
- Turbidity: not available

Details on source and properties of sediment:

- Site: Rhein at Mumpf (4332 Mumpf, Switzerland)
- Coordinates: 47.546024° N, 7.931460 °E
- Source of inoculum: sediment and water
- Sampling date: July 16, 2019
- Storage conditions: water-logged (>5 cm water layer) at about 4 °C in the dark
- Storage length: for up to 20 days until use
- Depth (sediment layer): 5-10
- Colour: greyisch brown
- Smell: slightly moldy

Duration of test (contact time):

177 d

Initial conc.:

15 µg/L

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Test temperature: 10.0 +/- 0.4 °C

SAMPLING
- Sampling frequency: duplicate samples of the Pond water/sediment system were taken immediately after application (0 h) and after 0.5, 1, 2, 3, 7, 14, 28, 60, 91, 120, 154, and 177 days of incubation.

Test performance:

Parameters during acclimatization: Adequate stability of all systems, as reflected by pH, oxygen concentration in water and redox potential of sediment and water, was reached during the two weeks acclimatisation period. Redox potentials and oxygen concentrations measured in the water phase of treated samples were indicative for aerobic conditions during the incubation period. Redox potentials in the sediment were indicative for anaerobic conditions. Similar values were found in the untreated controls, demonstrating that the test item had no significant effects on the physico-chemical parameters of the test system pH values of the water and sediment phase indicated stable conditions in both systems during the whole incubation period. The purity of [14C]BMDBM-MP was determined by HPLC to be 99.4% and 97.8% before and after application, and for [14C]BMDBM-TP in the application solution it was purity and stability of the test item during the application procedure. Total mean recoveries were 96.0 ± 3.1% (MP-label) and 95.3 ± 2.7% AR (TP-label) for Calwich Abbey water/sediment systems under aerobic conditions.

Compartment:

natural water / sediment

% Recovery:

96

St. dev.:

3.1

Remarks on result:

other: label 1 - [methoxyphenyl-ring-U-14C]BMDBM (MP-label)

Compartment:

natural water / sediment

% Recovery:

95.3

St. dev.:

2.9

Remarks on result:

other: label 2 - [tert-butyl-phenyl-ring-U-14C]BMDBM (TP-label)

Key result

Compartment:

natural water: freshwater

DT50:

1.4 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Pond water phase

Key result

Compartment:

natural sediment: freshwater

DT50:

139 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Pond sediment phase

Key result

Compartment:

natural water / sediment: freshwater

DT50:

99 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Pond water/sediment system

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

Up to 6 degradation products were detected in the pond water phase of label 1. Degradation products M1 and M3 reached maximum amounts of 1.9 % AR (DAT 60) and 0.9 % AR (DAT 28) in the water phase of label 1, respectively. The other minor degradation products did not exceed 0.4 % AR. In the sediment phase, BMDBM (label 1) increased from 25.5 % AR (DAT 0) to a maximum of 75.8 % AR (DAT 14) decreasing to 43.2 % AR (DAT 177). Two degradation products M1 and M3 were detected for label 1 amounting to a maximum of 2.4 % AR (DAT 91) and 6.9 % AR (DAT 120), respectively. In the total pond water-sediment system for label 1, M1 and M3 reached a maximum of 3.2 % AR and 7.1 % AR decreasing to 0.2 % AR and 4.4 % AR at the end of incubation, respectively. For label 2, up to 7 degradation products were detected in water samples. M2 represented the major degradation product detected at 2.1 % AR (DAT 7) to reach a maximum of 9.5 % AR (DAT 60) and amounting to 1.5 % AR on DAT 177. The other degradation products did not exceed 1.0 % AR (M11) throughout the study. In the sediment phase for label 2, three degradation products were detected. M2 reached a maximum of 2.8 % AR (DAT 60) and M3 was detected at 5.7 % AR (DAT 91). A third degradation product M11 was detected only once at 0.2 % AR. In the total pond water-sediment system for label 2, M2 and M3 were detected at a maximum of 12.4 % AR (DAT 60) and 5.7 % AR (DAT 91), respectively. At the end of incubation, M2 and M3 amounted to 3.9 % AR and 5.3 % AR, resppectively. The identity of M1 (label 1) and M2 (label 2) were confirmed by LC-MS analysis to be 4-methoxy benzoic acid (M1) and to be 4-tert-butylbenzoic acid (M2), respectively. M3 was detected for both labels. Based on the characterization by LC-MS/MS the proposed structure corresponds to 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione.

Evaporation of parent compound:

no

Volatile metabolites:

yes

Residues:

yes

Details on results:

Immediately after application (0 h), 70.7 % / 83.6 % (label 1 / 2) of the applied radioactivity was present in the water phase while 27.3 % / 13.1 % AR (label 1 / 2) were detected in the sediment. Throughout the study, the radioactivity in the water phase continuously decreased to 1.1 % / 4.1 % AR (label 1 / 2) on DAT 177. While the radioactivity in the sediment phase reached a maximum of 90.6 % AR on DAT 14 and 88.2 % AR on DAT 28 for label 1 and 2, respectively. At the end of incubation, 73.5 % AR and 63.5 % AR were detected in the sediment for label 1 and 2, respectively.
The extractable radioactivity from the sediment amounted to a maximum of 78.1 % / 79.0 % AR for label 1 / 2 on DAT 14 and 28, respectively. At the end of incubation 47.5 % / 44.4 % AR (label 1 / 2) was extractable from the sediment. Non-extractable radioactivity increased throughout the study reaching amounts of 26.0 % AR (label 1) and 19.2 % AR (label 2) at the end of incubation. An increasing amount of carbon dioxide was detected reaching 20.7 % / 24.0 % AR (label 1 / 2) at the end of incubation. Other volatile compounds did not exceed 0.2 % AR for any label.

Half-life of BMDBM in the pond water and sediment compartment is estimated based the on amount of BMDBM detected in the corresponding compartment for both labels. Calculation of the half-life of BMDBM in the sediment is estimated on data from sampling points starting with the maximum value for BMDBM from day 7 onwards till 177 DAT. SFO kinetic model was used for both the aqueous phase and sediment and Hockey Stick (HS) kinetic model was used for the total system. 

 

Compartment	DT50 (days) at 12 °C	DT90 (days)  at 12 °C
Pond water phase	1.4	4.5
Pond sediment phase	139	462
Pond water/sediment system	99	408

 

Validity criteria fulfilled:

yes

Conclusions:

Using a pond water/sediment system, the DT50 for the parent compound in the sediment phase was 139 days at 12 °C, for the pond water phase 1.4 days at 12 °C and for the entire pond water/sediment system, the DT50 was 99 days at 12 °C.

Executive summary:

The biodegradation of the test item in freshwater sediment was tested in a pond water/sediment according to OECD 308. The test item was labelled at 2 label positions - [methoxyphenyl-ring-U-14C]BMDBM (label 1) and [tert-butyl-phenyl-ring-U-14C]BMDBM (label 2). The radiolabelled BMDBM was applied onto the water phase of the equilibrated samples of the pond water / sediment system (Calwich Abbey Lake, England) at a target concentration of 15 µg/L. The treated water / sediment system was incubated at 10 ± 0.4 °C under aerobic conditions. Duplicate samples of the pond water/sediment system were taken immediately after application (0 h) and after 0.5, 1, 2, 3, 7, 14, 28, 60, 120, 154 and 177 days of incubation. Total recovery of the applied radioactivity was 96.0 ± 3.1 % AR (label 1) and 95.3 ± 2.9 % AR (label 2). In the pond system, immediately after application (0 h), 70.7 % / 83.6 % (label 1/2) of the applied radioactivity was present in the water phase. The mean amount of radioactivity extracted from sediments accounted for 25.5 % AR and 8.8 % AR at time 0 and increased over time to a maximum of 78.1 % AR (14 DAT) and 79.0 % AR (28 DAT), respectively for label 1 and 2. At study end, the extractable radioactivity from sediment represented 47.5 % AR for label 1 and 44.4 % AR for label 2. At time 0, mean amounts of non-extractable radioactivity accounted for 1.8 % (label 1) and 4.3 % AR (label 2). For both labels, formation of bound residues increased during incubation and reached maximum mean values of 32.3 % and 26.7 % AR, respectively, after 154 days. At study end, the non-extractable radioactivity from sediment represented 26.0 % for label 1 and 19.2 % for label 2. Considerable formation of [14]CO₂ was observed increasing throughout the study reaching 20.7 % and 24.0% AR in the lake system treated with label 1/2 at the end of incubation, respectively. Organic volatile products absorbed in the ethylene glycol traps did not exceed 0.3 % AR in any aquatic sediment system at any time. In the lake system, [methoxyphenyl-14C] and [tert-butyl-phenyl-14C]BMDBM dissipated rapidly from the aqueous phase within 14 DAT amounting to 70.4 % and 83.3 % AR immediately after treatment (mean) and to 3.2 % and 3.3 % AR on 14 DAT, thereafter it decreased further accounting for 0.7 % and 0.9 % AR on 177 DAT for label 1 and 2, respectively. Metabolites 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) peaked at 1.9 % and 9.5 % AR on 60 DAT, respectively. In addition, up to 6 minor metabolites were detected including M3 not exceeding 1.7 % AR at any time during the study. In the sediment extracts, the [methoxyphenyl-14C] and [tert-butyl-phenyl-14C]BMDBM amounted to 25.5 % and 8.6 % AR immediately after treatment and were detected at a maximum of 75.8 % and 79.0 % AR (14 and 28 DAT), thereafter decreasing to 43.2 % and 36.7 % AR at the end of incubation, respectively. Additionally, a total of five metabolites were formed. Besides the major metabolite 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reaching a maximum of 6.9 % AR, the two metabolites 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) reached maximum amounts of 2.4 % and 2.8 % AR, respectively. One additional unknown minor metabolite, was detected only once at a minor amount of < 1 %. With regard to the total lake system, [methoxyphenyl-14C] and [tert-butyl-phenyl-14C]BMDBM decreased from initially 95.9 % and 91.9 % AR (mean, time 0) to 43.9 % and 37.6 % respectively, by the end of incubation. In the lable 1 samples, 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reached up to 7.1 % AR on 120 DAT, and decreased by the end of incubation to 4.4 % AR. In the label 2 samples, metabolite M3 reached mean values of up to 5.7 % AR on 91 DAT and subsequently decreased to 5.3 % AR by the end of incubation. Furthermore, for the label 1, 4-methoxy-benzoic acid (M1) peaked at 3.2 % AR on day 60 and decreased to 0.2 % AR at study end. In label 2, para-tert-butylbenzoic acid (M2) peaked at 12.4 % AR on 60 DAT decreasing to 3.9 % AR at study end. In addition, up to four unknown metabolites were detected for both labels, one of them reaching a maximum of 1.7% AR, while none of the others were exceeding 0.4 % AR during the incubation time. Calculation of the half-life of BMDBM in the sediment is estimated on data from sampling points starting with the maximum value for BMDBM from DAT 7 onwards to DAT 177. SFO kinetic model was used for both the aqueous phase and sediment and Hockey Stick (HS) kinetic model was used for the total system. As a result, the DT50 for the parent compound in the sediment phase was 139 days at 12 °C, for the pond water phase 1.4 days at 12 °C and for the entire pond water/sediment system, the DT50 was 99 days at 12 °C.

Description of key information

Ultimate degradation in surface water (OECD 309)

The half-life, DT50, of the parent compound in natural water at 12 °C was determined to be 10.8 days at high dose (0.015 mg/L) and 10.2 days at low dose (0.005 mg/L). Under sterile conditions the parent compound degraded slower than in a non-sterile conditions. The half-life at 12 °C was 54.7 days at a concentration of 0.015 mg/L. The parent compound degraded to two major metabolites, depending on the placement of the radiolabel and a common metabolite for both labels.

 

Biodegradation in sediment (OECD 308)

The half-life, DT50, of the parent compound in freshwater sediment was determined to be 111 days at 12 °C in a river water/sediment system. The half-life in the water phase was 0.13 days at 12 °C and from the entire river water/sediment system the DT50 was determined to be 72.1 days at 12 °C.

Using a pond water/sediment system, the DT50 for the parent compound in the sediment phase was 139 days at 12 °C, for the pond water phase 1.4 days at 12 °C and for the entire pond water/sediment system, the DT50 was 99 days at 12 °C.

Key value for chemical safety assessment

Half-life in freshwater:

10.8 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

139 d

at the temperature of:

12 °C

Additional information

Comparative assessment of degradation in water systems

The route and rate of degradation of [¹⁴C]BMDBM was investigated in two different aquatic systems (an European river and pond) under aerobic conditions at 10.0 ± 0.4 °C. Additionally, aerobic mineralisation of [¹⁴C] BMDBM in surface water was investigated under defined laboratory conditions in the dark at 10.8 ± 0.8 °C at a high and a low dose and a high dosed sterile system to investigate the part of abiotic degradation.

The degradation of [14C]BMDBM in the surface water for the high dose and the low dose was comparable (11.9 d for the high dose and 11.2 d for the low dose), indicating that BMDBM degradation in surface water - is at least at the tested level - concentration-independent. The degradation in the surface water could adequately be described by single first order kinetics. The comparison of the degradation rates derived for the sterile system (DT₅₀ 60.2 d) and the biological active system (11.9 d) shows, that the main part of degradation is biotic. Biotic (degradation was calculated by subtracting the degradation constant obtained with the sterile samples from the one obtained for the high-dosed non-sterile samples with a DT₅₀ of 14.7 d at study temperature.

The results of the OECD 309 study with a degradation half-life for BMDBM of 11.9 d in surface water at 10°C  (corresponds to 10.8 d normalised to 12 °C according to FOCUS formular), demonstrate that BMDBM is not persistent in surface water according to the REACH criteria for persistency.

In the water-sediment study [14C]BMDBM rapidly dissipated from the water phase into the sediment resulting to half-lives of 0.13  d (river) and 1.4 d (pond) (corrected to 12 °C in FOCUS) in the water phase. With the dissipation from water phase, the part of radioactivity as NERs is steadily increasing in both systems from 0.6/1.9 to 27.8/30.7 % AR in river and 4.8/1.8% AR to 19.2/26.0 % AR in the pond system. However, while a part of radioactivity is transferred to the NER and sediment, the degradation in water is fast, as considerable mineralization could be shown by the increasing CO₂ in all systems, reaching 20.7 % and 24.0 % AR in the lake system treated with the MP- and TP-labels at the end of incubation, respectively and in the river system treated with the MP- and TP-labels, respectively, reaching 27.2 % and 19.9 % AR at the end of incubation.  

In both water-sediment systems, the dissipation process from the aqueous phase can be described by bi-phasic model with a very rapid dissipation from the water phase followed by a slower but still fast dissipation in the second phase. For both systems, similar DT₉₀ values of 8.4 d and 4.5 d (corrected to 12 °C in FOCUS) for the water phase were obtained, indicating comparable dissipation from the water phase in the medium term.  Under consideration of the results from aerobic degradation in surface water, dissipation of [14C]BMDBM appears to be a result of both fast degradation in the water phase and simultaneous phase transfer processes into the sediment. Regarding the slower degradation rates from the surface water system compared to the dissipation rates derived for the aqueous phase in the water-sediment systems transfer into the sediment seems to be the more pronounced of both fast processes.

Until DAT 7, the residues in sediment are constantly increasing. The degradation in the sediment phase is assessed via degradation kinetics from maximum onwards starting from DAT 7 with a single first order model, resulting in a DT50 of 130 d (river system) and 163 d (pond system) at 10°C, corresponding to 111d and 139 d at 12°C in river and pond system, respectively (temperature correction according to FOCUS). In view of the continuously fast dissipation of BMDBM from the water phase, it may be expected that transfer into sediment was still ongoing after the peak was reached on DAT 7. As the kinetic assessment does not consider the simultaneous processes of BMDBM transfer into the sediment and its degradation therein, the degradation rate is underestimated and actual half-lives may be expected to be lower than the indicated values.

The part of non-extractable residues (NER) increased during the study to 30.7/27.8 % (DAT 177 methoxyphenyl-¹⁴C / tert-butyl-phenyl-¹⁴C label) in the river system and 32.3/26.7 % (DAT 154 methoxyphenyl-¹⁴C / tert-butyl-phenyl-¹⁴C label) in the pond system. The fractionation of the humic substance via solution at low and at high pH showed that only a part of the NER could be dissoluted again at a high pH (29.8 %/26 % of NER) or a low pH (28.4 %/30.8 % of NER) in the river system and at a high pH (15.2 %/18.6 % of NER) or a low pH (22.1 %/22.2 % of NER) in the pond system.

 

The results of the OECD 308 study with a degradation half-life for BMDBM of maximal 163 d in sediment at 10 °C corresponding to 139 d at 12 °C according to FOCUS, the results of the OECD 308 study demonstrate that BMDBM is not very persistent in sediment according to REACH, Annex XIII.

 

Metabolites:

In the surface water system BMDBM was degraded mainly to three major metabolites. Two of them are 4-methoxy-benzoic acid (M4), para-tert-butylbenzoic acid (M6). Based on LC-MS/MS data the third major metabolite M10 was   proposed to correspond to 3-(4-tert-butyl-phenyl)-3-hydroxy-1-(4-methoxy-phenyl)-propan-1-one (M10). M10 was detected at a maximum of 7.8 AR % on DAT 15 in the high dose, and up to 10.7 % (32 DAT) in the low dose samples and decreased throughout the study amounting to 3.0 % and 3.9 % on 60 DAT in the high and low dose samples, respectively. The most predominant metabolite in both non-sterile systems treated with MP-labelled BMDBM was M4 amounting to 8.8 % and 5.8 % AR in high dose and low dose samples on 15 DAT, respectively. M4 reached maximum amounts up to 18.4 % and 22.6 % AR on day 60 in high and low dose systems, respectively. In the surface water system treated with TP-labelled BMDBM, M6 was the most prominent metabolite reaching maximum amounts of up to 49.8 % and 50.5 % AR in high and low dose systems on 60 DAT. 

For M10, a DT₅₀ could be derived by assessment of degradation from maximum onwards for the high dosed system, resulting in a DT₅₀ of 16.2  days at 12 °C (SFO). Degradation observed in non-sterile samples resulted also in formation of higher amounts of radioactive carbon dioxide (6.5 % and 7.7 % in the high and in the low dosed system vs. 0.3 % in sterile samples) and higher number of metabolites (6 additional) when compared with sterile samples.

In the aqueous phase of the water sediment system from river Rhein, after dissipation into the sediment phase, degradation of BMDBM to several minor metabolites in the water phase was observed. As detected in the surface water system, metabolites 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) were also detected in the aqueous phase of the river system reaching only minor amounts at 3.0% and 4.8% AR .  

In the sediment, one major metabolite M3 (1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione) was formed slowly, reaching maximum amounts of 13.1 % AR (MP-label) and 14.8 % AR (TP-label) in the sediment extracts on 91 DAT. With regards to the total river system the major metabolite 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reached up to 13.8 % (MP-label) and 16.5 % AR (TP-label) on 91 DAT in the water-sediment system and decreased to 11.5 % and 9.2 % AR, respectively, by the end of incubation. In the total river water-sediment system, 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) reached a maximum of 5.5 % and 6.6 % AR, respectively. By the end of incubation M1 and M2 decreased to 1.6 % and 2.9 % AR, respectively. In addition, formation of 14C-carbondioxide was observed throughout the study reaching 27.2 % and 19.9 % AR at the end of incubation in the river system treated with the MP- and TP-labels, respectively.

In the aqueous phase of the pond water-sediment system,  Metabolites 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) peaked at 1.9% and 9.5% AR on 60 DAT, respectively. Both metabolites decreased to 0.2 % and 1.5 % AR at the end of incubation, respectively. In addition, up to 6 minor metabolites were detected.

In the sediment extracts of the pond system, a total of five metabolites were formed. Besides the major metabolite 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reaching a maximum of 6.9% AR (MP-label, 120 DAT), the two metabolites 4-methoxy-benzoic acid (M1) and para-tert-butylbenzoic acid (M2) reached maximum amounts of 2.4 % and 2.8 % AR, respectively.

With regard to the total pond system, the metabolite 1-(4-tert-butylphenyl)-3-(4-hydroxyphenyl) propane-1,3-dione (M3) reached its maximum up to 7.1 % AR on 120 DAT in the MP-labelled samples. Furthermore, for the MP-label, 4-methoxy-benzoic acid (M1) peaked at 3.2 % AR on day 60 and decreased to 0.2 % AR at study end. In the TP-label, para-tert-butylbenzoic acid (M2) peaked at 12.4 % AR on 60 DAT decreasing to 3.9 % AR at study end (177 DAT). In addition, considerable formation of 14C-carbondioxide was observed throughout the study reaching 20.7 % and 24.0 % AR in the lake system treated with the MP- and TP-labels at the end of incubation, respectively.  

In both water sediment systems M3 was found to be only slightly declining towards the end of the study suggesting it was constantly formed and subsequently bound to the organic matter of the sediment. A DT-50 could not be derived for M3 due to its flat decline in the sediment.