Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr 2017 - 03 Nov 2017

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)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): natural aerobic surface water source in Rhineland-Palatinate (67374 Hanhofen, Germany, N49.312358, E8.318905). Water was sampled from the top 5 to 10 cm of the natural resource.
- Storage conditions: water was transported in polyethylene containers to the laboratory.
- Storage length: 1 day
- Temperature (°C) at time of collection: 14ºC
- pH at time of collection: 8.4
- Redox potential (mv) initial: 153
- Oxygen concentration (mg/l) initial: 12.3
- Dissolved organic carbon (mg/l): 9.3
- Water filtered: yes
- Type and size of filter used, if any: 100 μm mesh size.

Details on source and properties of sediment:

Not applicable

Details on inoculum:

Not applicable

Duration of test (contact time):

59 d

Initial conc.:

9.7 µg/L

Based on:

test mat.

Initial conc.:

91.3 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 500 mL surface water. 79 μL for FT flasks (9.7 μg/L). 107 μL for FT flasks (91.3 μg/L). 116 μL for FC flasks (21 μg/L). FC and FB flasks were additionally treated with 107 μL acetonitrile.
- Composition of medium: Fresh sampled water, biologically active
- Additional substrate: No
- Solubilising agent (type and concentration if used): No
- Test temperature: 12 ± 2
- pH: 7.96 (at study start)
- pH adjusted: no
- Aeration of dilution water: aeration was performed using slightly constant orbital movement and constant air flow.
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: None

TEST SYSTEM
- Culturing apparatus: Glass flasks (1000 mL)
- Number of culture flasks/concentration:
18 flasks, treated with 9.7 μg/L test item. 16 were analysed (2 flasks as reserve).
18 flasks, treated with 91.3 μg/L test item. 16 were analysed (2 flasks as reserve).
6 sterile samples, treated with 91.3 μg/L test item. 2 were analysed (4 flasks as reserve).
8 reference samples, treated with 21 μg/L reference substance and 107 μL of solvent, 8 were analysed (0 flasks as reserve)
2 blank controls, treated with 107 μL of solvent
- Method used to create aerobic conditions: slightly constant orbital movement and constant air flow.
- Method used to control oxygen conditions: dissolved oxygen concentration of the water phase was measured in the blank controls once a week throughout the study.
- Measuring equipment: see details of analytical methods.
- Details of trap for CO2 and volatile organics if used:
Trap for CO2: soda lime (8 g) trap (Sigma-Aldrich, 72073-1KG; ProLabo, 22666.362)
Trap for volatile organics: 2 polyurethan (PU) plug + 1 TenaxTM trap (TenaxTM Tubes Orbo 402, Supelco, 20832-U; TenaxTM TA 60/80 Porous Polymer Adsorbent, Supelco, 11982)

SAMPLING
- Sampling frequency: 0, 2, 7, 14, 21, 29, 45 and 59 days after treatment (DAT).
- Sampling method used per analysis type:
Organic Volatiles: Traps for organic volatiles (Polyurethane plug; PU) were extracted with 60 mL acetone for one hour on a flatbed shaker and the amount of radioactivity in the extract was determined by LSC of an aliquot (1 x 1 mL). TenaxTM as trap for organic volatiles was additionally used. The TenaxTM traps were extracted with 10 mL acetone for one hour on a flatbed shaker and the amount of radioactivity in the extract was determined by LSC of an aliquot (1 x 0.5 mL).
Carbon dioxide: Radioactive carbon dioxide, formed in the FT and FS flasks, was captured by soda lime (8 g) trap and was monitored at each sampling time. The radioactivity in soda lime was extracted with 60 mL hydrochloric acid (18%) in a closed system. The evolving 14CO2 was trapped by a scintillation cocktail oxysolve. The radioactivity in the extract was determined by LSC after dissolving. Dissolved 14CO2 was determined indirectly by stripping it out from the water phase. For this purpose 100 μL hydrochloric acid was added to a 1 mL aliquot of the water phase, which was shaken vigorously and incubated for an hour. The remaining radioactivity was measured by LSC.
Water phase: The total amount of radioactivity in the water phase was analysed in a 1 ml aliquot of the water phase by LSC. The radioactivity was analysed by LSC and HPLC.
pH-value and Oxygen Concentration: The pH-value and the dissolved oxygen concentration of the water phase were measured in the blank controls once a week throughout the study.
- Sample storage before analysis: The samples were analysed immediately or stored at ≤ -18 °C. Thereafter the samples were stored in a freezer at ≤ -18 °C

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 blank controls, treated with 107 μL of solvent
- Abiotic sterile control: 6 sterile samples, treated with 91.3 μg/L test item. 2 were analysed at day 29 of the incubation (4 flasks as reserve). Sterilized test flasks were prepared by autoclaving for 20 min at 121ºC.

STATISTICAL METHODS:
The disappearance time (DT) of the test substance was calculated after analysis of the last sampling, including information about the dissipation/degradation kinetics according to the recommendations of EC document Sanco/10058/2005 version 2.0.
The calculation of the rate constant and the initial concentration was performed using the software CAKE 3.3 (2016). The initial concentration at 0 d was included in the parameter optimization procedure, but for an optimal fit, the value was allowed to be estimated by the model. For the evaluation of the data three different kinetic models (single first order, double first order in parallel, first order multi compartment) were tested in order to find the most suitable approach based on the Prob>t criterion and visual assessment.

Reference substance:

benzoic acid, sodium salt

Remarks:

([ring-U-14C]Benzoic acid, sodium salt; radiolabelled reference substance)

Compartment:

natural water: freshwater

% Recovery:

86.8

Remarks on result:

other: Low tested concentration at the end of experiment

Compartment:

natural water: freshwater

% Recovery:

87.5

Remarks on result:

other: High tested concentration at the end of experiment. Due to a divergent HPLC pattern only values of one sample was used for evaluation

Key result

% Degr.:

94.9

Parameter:

radiochem. meas.

Sampling time:

59 d

Remarks on result:

other: Low tested concentration

Key result

% Degr.:

43.8

Parameter:

radiochem. meas.

Sampling time:

59 d

Remarks on result:

other: High tested concentration. Due to a divergent HPLC pattern only values of one sample was used for evaluation

Key result

Compartment:

natural water: freshwater

DT50:

24.6 d

Type:

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

Temp.:

11.7 °C

Remarks on result:

other: Low tested concentration

Key result

Compartment:

natural water: freshwater

DT50:

117 d

Type:

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

Temp.:

11.7 °C

Remarks on result:

other: High tested concentration

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#1

No.:

#4

No.:

#4

No.:

#6

Details on transformation products:

- Formation and decline of each transformation product during test:
Compounds (4-tert-butyl-2,5-dimethylphenyl) sulfonic acid (M1), M4, M6 and M2 were identified as major metabolites of the test item. M1 reached maximum values of 36.1 % AR (59 days after treatment) and 16.6 % AR (59 days after treatment) for the low
and high test concentration, respectively.
Metabolite M4 was observed in amounts up to 19.6% AR (low concentration) and 1.7 % AR (high concentration) at 59 and 21 DAT, respectively. The metabolite M6 was only detected in the samples of the low concentration in maximum amounts of 7.0 % AR after 59 days of treatment. The metabolite M2 was only observed in the high concentrated samples in a maximum value of 9.6 % AR after 59 days of treatment. This metabolite could not be identified by LC-MS.
Six unknown peaks (M2, M3, M7, M8, M9 and M14) in amounts <5 % AR were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M5, M10, M11, M12 and M13) were detected in amounts <5 % AR.
- Pathways for transformation: see attached picture

Evaporation of parent compound:

no

Volatile metabolites:

no

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): No

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Low test concentration:
M1: 36.1% AR in 45 DAT; M4: 19.6% AR in 59 DAT and M6: 7% AR in 59 DAT.
High test concentration:
M1: 16.6% AR in 59 DAT and M2: 9.6% AR in 59 DAT.
- Range of maximum concentrations in % of the applied amount at end of study period:
M1: 33.3% AR; M4: 19.6% AR and M6: 7%.
High test concentration:
M1: 16.6% AR and M2: 9.6% AR.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Low test concentration:
M2: 0.9% AR in 29 DAT; M3: 2.4% AR in 2 DAT; M7: 4.7% AR in 59 DAT; M8: 1.7% AR in 59 DAT; M9: 1.2% AR in 59 DAT and M14: 1.7% AR in 59 DAT
High test concentration:
M5: 3% AR in 2 and 14 DAT; M4: 1.7% AR in 21 DAT; M10: 1.5% AR in 29 DAT; M11: 1.4% AR in 21 DAT; M12: 1.1% AR in 21 DAT and M13: 1.2% AR in 29 DAT
- Range of maximum concentrations in % of the applied amount at end of study period:
Low test concentration:
M2: 0% AR; M3: 1.2% AR; M7: 4.7% AR; M8: 1.7% AR; M9: 1.2% AR and M14: 1.7% AR
High test concentration:
M5: 0% AR; M4: 0% AR; M10: 0% AR; M11: 0% AR; M12: 0% AR and M13: 0% AR

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
Low test concentration: 13.2% AR
High test concentration: 12.5% AR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:
Low test concentration: 7.9% AR
High test concentration: 1.5% AR

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:
Low test concentration: 3.3% AR
High test concentration: 3.7% AR

STERILE TREATMENTS (if used)
- Transformation of the parent compound: 1.3% AR of CO2 + 1.9% AR of organic volatiles + 2.1% of metabolite in 29 DAT (end of study)
- Formation of transformation products: M1, 2.1% AR in 29 DAT (end of study)
- Volatilization: 1.9% AR

Results with reference substance:

The test samples containing the reference item were sampled and analysed at day 0 and 10. The recovery of radioactivity in the water system of the reference samples was 94.3 % and 93.1 % AR at 0 and 10 DAT, respectively. After 10 days 82.5 % AR of the reference item was mineralised. This indicates that the test system was biologically active. Therefore, the test was valid.

Table 1: Distribution of the radioactivity between test water, carbon dioxide and organic volatiles in % of the applied radioactivity (AR) for the 2,5-Dimethyl-4-tertbutyl[U-14C]phenol test concentration of 9.7 µg/L

FT10-samples
Sampling Interval [d]	0	2	7	14	21	29	45	59
	% AR
Water phase	99.0	96.4	94.9	93.0	89.0	83.6	87.5	77.7
	99.6	97.0	95.6	91.0	92.7	80.9	76.7*	73.5
Mean	99.3	96.7	95.2	92.0	90.8	82.3	82.1	75.6
Total carbon dioxide	1.4	0.4	1.0	1.6	7.8	5.0	0.6	8.2
	0	0.3	0	1.0	0.1	9.2	9.1*	7.6
Mean	0.7	0.3	0.5	1.3	3.9	7.1	4.8	7.9
Organic volatiles	n.a.	0.4	0.9	1.2	2.6	2.4	3.5	3.5
	n.a.	0.5	0.9	1.2	2.0	1.8	3.0*	3.1
Mean	n.a.	0.4	0.9	1.2	2.3	2.1	3.2	3.2
Recovery	100.4	97.2	96.8	95.9	99.4	91.1	91.6	89.3
	99.6	97.8	96.5	93.3	94.7	92.0	88.8*	84.3
Mean	100.0	97.5	96.7	94.6	97.0	91.5	90.2	86.8

*: not used because mass balance < 90%

Table 2: Characterisation of the water phase in % of the applied radioactivity (AR) for the 2,5-Dimethyl-4-tertbutyl[U-14C]phenol test concentration of 9.7 µg/L

FT10-samples
Sampling interval [days]	0	2	7	14	21	29	45	59
	% AR
Parent	97.6	92.5	91.0	85.9	65.6	44.6	49.8	4.7
	99.0	92.5	91.7	73.4	69.1	41.2	6.2*	5.6
M1	0.9	0.8	1.7	3.5	15.9	21.3	24.4	35.7
	0.7	1.2	1.8	8.0	13.0	23.3	47.8*	30.9
M2	0.5	0	0	0	0	1.1	0	0
	0	0	0	0	0	0.6	0.6*	0
M3	0	2.3	0	0	0	0	0.5	1.4
	0	2.4	0	0	0.5	0.6	1.3*	1.0
M4	0	0.8	1.5	3.0	4.9	9.2	8.5	18.5
	0	0.8	1.6	5.7	7.0	8.2	11.1*	20.7
M6	0	0	0.6	0.6	1.5	3.4	2.2	7.0
	0	0	0.6	1.2	1.8	3.9	7.2*	7.0
M7	0	0	0	0	0.6	2.2	1.4	5.2
	0	0	0	0.6	0.9	1.8	0*	4.3
M8	0	0	0	0	0.5	0.9	0.7	1.9
	0	0	0	1.3	0.3	0.7	1.5*	1.6
M9	0	0	0	0	0	0.8	0	1.3
	0	0	0	0.8	0	0.7	1.0*	1.1
M14	0	0	0	0	0	0	0	2.0
	0	0	0	0	0	0	0*	1.4
Total	99.0	96.4	94.9	93.0	89.0	83.6	87.5	77.7
	99.6	97.0	95.6	91.0	92.7	80.9	76.7*	73.5

*: not evaluated, because the mass balance was < 90%

Table 3: Distribution of the radioactivity between test water, carbon dioxide and organic volatiles in % of the applied radioactivity (AR) for the 2,5-Dimethyl-4-tertbutyl[U-14C]phenol test concentration of 91.3 µg/L

FT95-samples
Sampling Interval	0	2	7	14	21	29	45	59
	% AR
Water phase	100.5	97.1	94.4	90.8	91.1	94.5	87.6	82.4
	99.4	94.3	94.7	91.9	93.0	91.4	92.0	80.3*
Mean	99.9	95.7	94.5	91.3	92.1	93.0	89.8	81.3
Total carbon dioxide	0	0	2.0	2.5	1.0	0.1	4.7	1.5
	0.2	0.4	1.3	0.8	0.1	2.3	0.1	3.1*
Mean	0.1	0.2	1.6	1.7	0.5	1.2	2.4	2.3
Organic volatiles	n.a.	0.2	0.7	1.4	2.4	1.5	1.7	3.7
	n.a.	0.3	0.7	1.3	1.7	1.2	2.6	3.5*
Mean	n.a.	0.3	0.7	1.3	2.0	1.4	2.2	3.6
Recovery	100.5	97.3	97.0	94.7	94.4	96.1	94.0	87.5
	99.5	95.0	96.6	94.0	94.8	94.9	94.8	86.9*
Mean	100.0	96.2	96.8	94.3	94.6	95.5	94.4	87.2

*: due to a divergent HPLC pattern this sample was not used for evaluation.

Table 4: Characterisation of the water phase in % of the applied radioactivity (AR) for the 2,5-Dimethyl-4-tertbutyl[U-14C]phenol test concentration of 91.3 µg/L

FT95-samples
Sampling interval [days]	0	2	7	14	21	29	45	59
	% AR
Parent	100.5	94.4	94.4	83.3	81.2	82.5	81.8	56.2
	99.4	91.1	94.7	86.9	81.8	79.7	86.7	0.0*
M5	0	2.7	0	0	0	0	0	0
	0	3.2	0	0	0	0	0	0*
M1	0	0	0	3.5	4.7	5.1	5.7	16.6
	0	0	0	2.5	3.8	5.4	5.3	56.5*
M4	0	0	0	1.4	1.5	1.7	0	0.0
	0	0	0	1.4	2.0	1.5	0	23.9*
M10	0	0	0	1.2	1.4	1.4	0	0
	0	0	0	1.1	1.3	1.6	0	0*
M11	0	0	0	1.4	1.4	1.0	0	0
	0	0	0	0	1.4	0.9	0	0*
M12	0	0	0	0	0.9	0	0	0
	0	0	0	0	1.2	0	0	0*
M2	0	0	0	0	0	1.5	0	9.6
	0	0	0	0	1.4	1.2	0	0*
M13	0	0	0	0	0	1.3	0	0
	0	0	0	0	0	1.1	0	0*
Total	100.5	97.1	94.4	90.8	91.1	94.5	87.6	82.4
	99.4	94.3	94.7	91.9	93.0	91.4	92.0	80.3*

*: due to a divergent HPLC pattern this sample was not used for evaluation.

Table 5: Complete results of the kinetic analysis in all tested water systems

Test water (applied concentration)	Best Fit Kinetic Model	DT50 [h]	DT90 [h]	Chi^2Error [%]	r^2 (observed vs. predicted)	Prob. > t**	Visual Assessment *
9.7	SFO	26.9	89.4	12.4	0.8986	1.91E-006	-
	DFOP	26.9	89.4	14.3	0.8986	0.5 / 0.5	-
	FOMC	21.2	70.6	13.2	0.8985	N/A	-
91.3	SFO	117	390	5.75	0.7053	6.97E-005	o
	DFOP	119	419	6.58	0.7279	0.4987 / 7.40E-004	o
	FOMC	118	392	6.14	0.7053	N/A	o

Best fit kinetic highlighted in bold letters (based on lowest chi2 error)

* Visual Assessment: + = good, o = moderate, - = poor

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

SFO: single first order; DFOP: double first order in parallel; FOMC: first order multi compartment

nd: not determined

N/A: not applicable

Validity criteria fulfilled:

yes

Conclusions:

In a simulation biodegradation test performed in aerobic natural surface water, degradation half-time DT50 values for 2,5-Dimethyl-4-tertbutyl[U-14C]phenol were 24.6 days (9.7 μg/L) and 117 days (91.3 μg/L).

Executive summary:

A simulation biodegradation test was conducted for radiolabelled test item 2,5-Dimethyl-4-tertbutyl[U-14C]phenol with two different application rates (9.7μg/L and 91.3μg/L) under aerobic conditions in the dark using natural aerobic surface water from the large water body Rhineland-Palatinate (67374 Hanhofen, Germany). The study was conducted in accordance with the OECD Guideline 309 and GLP. Duplicate samples were taken for analysis at specified intervals up to 59 days after application.Traps for organic volatiles and carbon dioxide were used.The radioactivity was quantified by liquid scintillation counting and characterised byHPLC. LC-MS was used for confirmation of metabolites in selected samples.

The recovery in the water system was in the range of 86.8 % to 100.0 % applied radioactivity (AR) with the low test concentration and 87.5 % to 100.0 % AR with the high test concentration.

CO2 was observed in amounts up to 7.9 % and 2.4 % AR for the low and high test concentration, respectively. Organic volatiles were detected in maximal amounts of 3.5 % AR (low concentration) and 3.7 % AR (high concentration).

Compounds (4-tert-butyl-2,5-dimethylphenyl) sulfonic acid (M1), 5-tert-butyl-2-hydroxy-4-methylbenzoic acid (M4), 4-tert-butyl-5-(hydroxymethyl)-2-methylphenyl hydrogen sulfate (M4), 4-tert-butyl-2-formyl-5-methylphenyl hydrogen sulfate (M6) and M2 were identified as major metabolites of the test item. M1 reached maximum values of 36.1 % AR (45 days after treatment, DAT) and 16.6 % AR (59 DAT) for the low and high test concentration, respectively. M4 was observed in amounts up to 19.6% AR (low concentration) and 1.7% AR (high concentration) at 59 and 21 DAT, respectively. M6 was only detected in the samples of the low concentration in maximum amounts of 7.0 % AR after 59 DAT. M2 was only observed in the high concentrated samples in a maximum value of 9.6 % AR after 59 DAT. This metabolite could not be identified by LC-MS.

Six unknown peaks (M2, M3, M7, M8, M9 and M14) in amounts <5 % AR were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M5, M10, M11, M12 and M13) were detected in amounts <5 % AR.

The test system was validated with the reference substance sodium benzoate. After 10 days 82.5 % AR of the reference item was mineralized. Thus, the system was biologically active.

The DT50 values for the test item were 24.6 days (9.7μg/L) and 117 days (91.3μg/L).

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:

24 Apr 2017 - 02 Aug 2017

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)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): natural aerobic surface water source in Rhineland-Palatinate (67374 Hanhofen, Germany, N49.312358, E8.318905). Water was sampled from the top 5 to 10 cm of the natural resource.
- Storage conditions: water was transported in polyethylene containers to the laboratory.
- Storage length: 1 day
- Temperature (°C) at time of collection: 14ºC
- pH at time of collection: 8.4
- Redox potential (mv) initial: 153
- Oxygen concentration (mg/l) initial: 12.3
- Dissolved organic carbon (mg/l): 9.3
- Water filtered: yes
- Type and size of filter used, if any: 100 μm mesh size.

Details on source and properties of sediment:

Not applicable

Details on inoculum:

Not applicable

Duration of test (contact time):

59 d

Initial conc.:

9.5 µg/L

Based on:

test mat.

Initial conc.:

92.9 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 500 mL surface water. 91 μL for FT flasks (9.5 μg/L). 107 μL for FT flasks (92.9 μg/L). 116 μL for FC flasks (21 μg/L). FC and FB flasks were additionally treated with 107 μL acetonitrile.
- Composition of medium: Fresh sampled water, biologically active
- Additional substrate: No
- Solubilising agent (type and concentration if used): No
- Test temperature: 12 ± 2
- pH: 7.96 (at study start)
- pH adjusted: no
- Aeration of dilution water: aeration was performed using slightly constant orbital movement and constant air flow.
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: None

TEST SYSTEM
- Culturing apparatus: Glass flasks (1000 mL)
- Number of culture flasks/concentration:
18 flasks, treated with 9.5 μg/L test item. 18 were analysed (0 flasks as reserve).
18 flasks, treated with 92.9 μg/L test item. 18 were analysed (0 flasks as reserve).
4 sterile samples, treated with 92.9 μg/L test item. 2 were analysed (2 flasks as reserve).
8 reference samples, treated with 21 μg/L reference substance and 107 μL of solvent, 8 were analysed (0 flasks as reserve)
2 blank controls, treated with 107 μL of solvent
- Method used to create aerobic conditions: slightly constant orbital movement and constant air flow.
- Method used to control oxygen conditions: dissolved oxygen concentration of the water phase was measured in the blank controls once a week throughout the study.
- Measuring equipment: see details of analytical methods.
- Details of trap for CO2 and volatile organics if used:
Trap for CO2: soda lime (8 g) trap (Sigma-Aldrich, 72073-1KG; ProLabo, 22666.362)
Trap for volatile organics: 2 polyurethan (PU) plug + 1 TenaxTM trap (TenaxTM Tubes Orbo 402, Supelco, 20832-U; TenaxTM TA 60/80 Porous Polymer Adsorbent, Supelco, 11982)

SAMPLING
- Sampling frequency: 0, 0.08, 0.17, 1, 7, 14, 29, 45 and 59 days after treatment (DAT).
- Sampling method used per analysis type:
Organic Volatiles: Traps for organic volatiles (Polyurethane plug; PU) were extracted with 60 mL acetone for one hour on a flatbed shaker and the amount of radioactivity in the extract was determined by LSC of an aliquot (1 x 1 mL). TenaxTM as trap for organic volatiles was additionally used. The TenaxTM traps were extracted with 10 mL acetone for one hour on a flatbed shaker and the amount of radioactivity in the extract was determined by LSC of an aliquot (1 x 1 mL).
Carbon dioxide: Radioactive carbon dioxide, formed in the FT and FS flasks, was captured by soda lime (8 g) trap and was monitored at each sampling time. The radioactivity in soda lime was extracted with 60 mL hydrochloric acid (18%) in a closed system. The evolving 14CO2 was trapped by a scintillation cocktail oxysolve. The radioactivity in the extract was determined by LSC after dissolving. Dissolved 14CO2 was determined indirectly by stripping it out from the water phase. For this purpose 100 μL hydrochloric acid was added to a 1 mL aliquot of the water phase, which was shaken vigorously and incubated for an hour. The remaining radioactivity was measured by LSC.
Water phase: The total amount of radioactivity in the water phase was analysed in a 1 ml aliquot of the water phase by LSC. The radioactivity was analysed by LSC and HPLC.
pH-value and Oxygen Concentration: The pH-value and the dissolved oxygen concentration of the water phase were measured in the blank controls once a week throughout the study.
- Sample storage before analysis: The samples were analysed immediately or stored at ≤ -18 °C. Thereafter the samples were stored in a freezer at ≤ -18 °C

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 blank controls, treated with 107 μL of solvent
- Abiotic sterile control: 4 sterile samples, treated with 92.9 μg/L test item. 2 were analysed at day 14 of the incubation (2 flasks as reserve). Sterilized test flasks were prepared by autoclaving for 20 min at 121ºC.

STATISTICAL METHODS:
The disappearance time (DT) of the test substance was calculated after analysis of the last sampling, including information about the dissipation/degradation kinetics according to the recommendations of EC document Sanco/10058/2005 version 2.0.
The calculation of the rate constant and the initial concentration was performed using the software CAKE 3.3 (2016). The initial concentration at 0 d was included in the parameter optimization procedure, but for an optimal fit, the value was allowed to be estimated by the model. For the evaluation of the data three different kinetic models (single first order, double first order in parallel, first order multi compartment) were tested in order to find the most suitable approach based on the Prob>t criterion and visual assessment.

Reference substance:

benzoic acid, sodium salt

Remarks:

([ring-U-14C]Benzoic acid, sodium salt; radiolabelled reference substance)

Compartment:

natural water: freshwater

% Recovery:

95.4

Remarks on result:

other: Low tested concentration at the end of experiment. Only single value was used due to mass balance < 90%.

Compartment:

natural water: freshwater

% Recovery:

92.5

Remarks on result:

other: High tested concentration at the end of experiment.

Key result

% Degr.:

99.1

Parameter:

radiochem. meas.

Sampling time:

59 d

Remarks on result:

other: Low tested concentration. Only single value was used due to mass balance < 90%

Key result

% Degr.:

88

Parameter:

radiochem. meas.

Sampling time:

59 d

Remarks on result:

other: High tested concentration.

Key result

Compartment:

natural water: freshwater

DT50:

11.5 d

Type:

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

Temp.:

11.7 °C

Remarks on result:

other: Low tested concentration

Key result

Compartment:

natural water: freshwater

DT50:

18.3 d

Type:

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

Temp.:

11.7 °C

Remarks on result:

other: High tested concentration

Other kinetic parameters:

first order rate constant

pseudo-first order rate constant

Transformation products:

yes

No.:

#5

No.:

#6

Details on transformation products:

- Formation and decline of each transformation product during test:
The compounds 2-tert-butyl-4-hydroxy-4,6-dimethylcyclohexa-2,5-dien-1-one (M5) and 3-tert-butyl-4-hydroxy-5-methylbenzoic acid (M6) were identified as major metabolites of the test item. M5 reached maximum values of 53.8 % AR (59 DAT) and 42.5 % AR (59 DAT) for the low and high test concentration, respectively. M6 was observed in amounts up to 16.7 % AR (low concentration) and 17.1 % AR (high concentration) at 59 DAT. Seven unknown peaks (M1, M2, M3, M4, M7, M8 and M12) in amounts <5 % AR (except M8 with 5.2 % AR at 29 DAT) were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M8, M9, M10, M11 and M13) were detected in amounts <5 % AR.
- Pathways for transformation: see attached picture

Evaporation of parent compound:

no

Volatile metabolites:

no

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): No

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Low test concentration:
M5: 53.8% AR in 59 DAT and M6: 16.7% AR in 59 DAT.
High test concentration:
M5: 42.5% AR in 59 DAT and M6: 17.1% AR in 59 DAT.
- Range of maximum concentrations in % of the applied amount at end of study period:
M5: 53.8% AR and M6: 16.7% AR.
High test concentration:
M5: 42.5% AR and M6: 17.1% AR.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Low test concentration:
M1: 2.4% AR in 0 DAT; M2: 3% AR in 0.17 DAT; M3: 0.6% AR in 0 DAT; M4: 3.2% AR in 29 DAT; M7: 1.1% AR in 29 DAT; M8: 5.2% AR in 29 DAT and M12: 2.3% AR in 45 DAT
High test concentration:
M8: 2.9% AR in 29 DAT; M9: 4.3% AR in 45 DAT; M10: 1.8% AR in 14 DAT; M11: 0.9% AR in 29 DAT and M13: 2.4% AR in 45 DAT
- Range of maximum concentrations in % of the applied amount at end of study period:
Low test concentration:
M1: 1.3%; M2: 0%; M3: 0%; M4: 2.6%; M7: 0%; M8: 2.9% and M12: 1.5%
High test concentration:
M8: 0%; M9: 0%; M10: 0%; M11: 0% and M13: 0%

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
Low test concentration: 4.6% AR
High test concentration: 7.5% AR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:
Low test concentration: 2% AR
High test concentration: 5% AR

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:
Low test concentration: 13.6% AR
High test concentration: 16.7% AR

STERILE TREATMENTS (if used)
- Transformation of the parent compound: 1.1% AR of CO2 + 11.8% AR of organic volatiles + 14.8% of metabolites in 14 DAT (end of study)
- Formation of transformation products: M9, 2.8% AR+ M5, 3.4% AR + M8, 8.6% in 14 DAT (end of study)
- Volatilization: 1.9% AR

Results with reference substance:

The test samples containing the reference item were sampled and analysed at day 0 and 10. The recovery of radioactivity in the water system of the reference samples was 94.3 % and 93.1 % AR at 0 and 10 DAT, respectively. After 10 days 82.5 % AR of the reference item was mineralised. This indicates that the test system was biologically active. Therefore, the test was valid.

Table 1: Distribution of the radioactivity between test water, carbon dioxide and organic volatiles in % of the applied radioactivity (AR) for the 2,4-Dimethyl-6-tertbutyl[U-14C]phenol test concentration of 9.5 µg/L

FT10-samples
Sampling Interval [days]	0	0.08	0.17	1	7	14	29	45	59
	% AR
Water phase	90.3	96.0	96.4	93.0	88.2	86.2	75.3*	80.0	71.5
	96.1	100.3	95.4	93.6	87.5	86.9	78.1	76.6	79.8*
Mean	93.2	98.1	95.9	93.3	87.8	86.5	76.7	78.3	75.7
Total carbon dioxide	12.1	1.1	0.7	4.1	3.2	2.4	1.7*	0.1	1.9
	1.5	1.4	3.7	3.7	2.9	0.8	1.5	4.3	2.0*
Mean	6.8	1.2	2.2	3.9	3.1	1.6	1.6	2.2	2.0
Organic volatiles	n.a.	0.7	0.5	1.5	8.7	12.6	14.0*	12.0	8.9
	n.a.	0.6	0.7	1.0	7.4	11.7	5.4	11.0	13.6*
Mean	n.a.	0.7	0.6	1.2	8.0	12.1	9.7	11.5	11.2
Recovery	102.4	97.7	97.7	98.6	100.1	101.2	91.0*	92.1	82.4
	97.6	102.4	99.9	98.3	97.8	99.3	84.9	91.9	95.4*
Mean	100.0	100.0	98.8	98.4	98.9	100.3	87.9	92.0	88.9

*only single value was used due to mass balance < 90%

Table 2: Characterisation of the water phase in % of the applied radioactivity (AR) for the 2,4-Dimethyl-6-tertbutyl[U-14C]phenol test concentration of 9.5 µg/L

FT10-samples
Sampling interval [days]	0	0.08	0.17	1	7	14	29	45	59
	% AR
Parent	89.5	96.0	96.4	89.7	63.0	39.0	16.4	2.5	0
	93.1	94.9	89.5	90.6	61.7	43.8	12.2	2.8	0.9
M1	3.8	0	0	0	0	0.6	0	1.1	1.4
	1.1	0	0	0	0	0.4	1.2	0.8	1.3
M2	0	0	0	0	0	1.4	0	0	0
	0.7	0	5.9	0	0	0	0	0	0
M3	0	0	0	0	0	0	0	0	0
	1.2	0	0	0	0	0	0	0	0
M4	0	0	0	0	0	1.9	3.2	2.4	4.0
	0	4.0	0	0	1.6	1.2	1.7	2.6	2.6
M5	0	0	0	2.3	17.1	28.6	38.1	46.6	47.9
	0	1.4	0	1.8	17.1	31.4	49.1	49.9	53.8
M6	0	0	0	1.1	4.6	12.2	11.4	18.4	13.3
	0	0	0	1.1	5.7	7.3	8.8	13.7	16.7
M7	0	0	0	0	1.5	0	1.1	1.7	0
	0	0	0	0	0	0	0	0	0
M8	0	0	0	0	1.9	2.6	5.2	4.8	3.4
	0	0	0	0	1.4	2.8	5.1	4.9	2.9
M12	0	0	0	0	0	0	0	2.5	1.6
	0	0	0	0	0	0	0	2.0	1.5
Total	90.3	96.0	96.4	93.0	88.2	86.2	75.3	80.0	71.5
	96.1	100.3	95.4	93.6	87.5	86.8	78.1	76.6	79.8

Table 3: Distribution of the radioactivity between test water, carbon dioxide and organic volatiles in % of the applied radioactivity (AR) for the 2,4-Dimethyl-6-tertbutyl[U-14C]phenol test concentration of 92.9 µg/L

FT95-samples
Sampling Interval [days]	0	0.08	0.17	1	7	14	29	45	59
	% AR
Water phase	95.5	94.0	92.6	88.3	84.4	81.0	69.2	73.3	71.5
	98.2	92.9	92.3	89.0	84.5	79.0	76.8	69.8	70.0
Mean	96.8	93.4	92.5	88.7	84.5	80.0	73.0	71.6	70.8
Total carbon dioxide	2.9	3.0	0.7	5.2	3.0	0.3	1.6	4.2	4.5
	3.5	2.4	1.2	5.3	2.9	2.0	0.9	4.4	5.6
Mean	3.2	2.7	0.9	5.2	3.0	1.1	1.3	4.3	5.0
Organic volatiles	n.a.	1.0	1.1	1.6	7.0	10.1	17.8	15.2	17.8
	n.a.	0.5	0.6	1.3	8.1	12.5	10.1	18.6	15.6
Mean	n.a.	0.7	0.8	1.5	7.6	11.3	13.9	16.9	16.7
Recovery	98.3	98.0	94.4	95.1	94.5	91.4	88.6*	92.8	93.8
	101.7	95.8	94.1	95.6	95.5	93.5	87.8*	92.9	91.2
Mean	100.0	96.9	94.2	95.3	95.0	92.4	88.2	92.8	92.5

*: the water phases of both samples were used for evaluation

Table 4: Characterisation of the water phase in % of the applied radioactivity (AR) for the 2,4-Dimethyl-6-tertbutyl[U-14C]phenol test concentration of 92.9 µg/L

FT95-samples
Sampling interval [days]	0	0.08	0.17	1	7	14	29	45	59
	% AR
Parent	95.5	94.0	92.6	88.3	73.0	55.1	26.9	20.3	9.8
	98.2	92.9	92.3	89.0	72.9	53.1	38.0	20.2	14.2
M9	0	0	0	0	1.8	0	0	0	0
	0	0	0	0	1.5	0	0	8.5	0
M6	0	0	0	0	3.6	7.3	7.2	10.2	15.1
	0	0	0	0	1.6	4.7	9.1	6.5	19.1
M5	0	0	0	0	6.0	15.4	28.8	34.2	48.4
	0	0	0	0	5.1	17.7	25.5	34.6	36.5
M8	0	0	0	0	0	1.6	3.3	3.9	0
	0	0	0	0	3.4	1.6	2.5	0	0
M10	0	0	0	0	0	1.7	1.2	0	0
	0	0	0	0	0	1.9	1.8	0	0
M11	0	0	0	0	0	0	1.9	0	0
	0	0	0	0	0	0	0	0	0
M13	0	0	0	0	0	0	0	4.7	0
	0	0	0	0	0	0	0	0	0
Total	95.5	94.0	92.6	88.3	84.4	81.0	69.2*	73.3	71.5
	98.2	92.9	92.3	89.0	84.5	79.0	76.8*	69.9	70.0

*: the water phases of both samples were used for evaluation

Table 5: Complete results of the kinetic analysis in all tested water systems

Test water (applied concentration)	Best Fit Kinetic Model	DT50 [h]	DT90 [h]	Chi^2Error [%]	r^2 (observed vs. predicted)	Prob. > t** k1_Parent/k2_Parent	Visual Assessment *
9.5	SFO	11.5	38.1	3.14	0.9939	2.51E-012	o
	DFOP	11.5	38.1	3.54	0.9939	0.4997 / 0.5	o
	FOMC	11.5	38.1	3.32	0.9939	N/A	o
92.9	SFO	19.1	63.6	2.06	0.9933	1.84E-015	o
	DFOP	18.3	68.7	1.91	0.994	0.3183 / 0.1658	o
	FOMC	18.3	69.5	1.8	0.994	N/A	o

Best fit kinetic highlighted in bold letters (based on lowest chi2 error)

* Visual Assessment: + = good, o = moderate, - = poor

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

SFO: single first order; DFOP: double first order in parallel; FOMC: first order multi compartment

nd: not determined

N/A: not applicable

Validity criteria fulfilled:

yes

Conclusions:

In a simulation biodegradation test performed in aerobic natural surface water, degradation half-time DT50 values for 2,4-Dimethyl-6-tertbutyl[U-14C]phenol were 11.5 days (9.5 μg/L) and 18.3 days (92.9 μg/L).

Executive summary:

A simulation biodegradation test was conducted for radiolabelled test item 2,4-Dimethyl-6-tertbutyl[U-14C]phenol with two different application rates (9.5 μg/L and 92.9 μg/L) under aerobic conditions in the dark using natural aerobic surface water from the large water body Rhineland-Palatinate (67374 Hanhofen, Germany). The study was conducted in accordance with the OECD Guideline 309 and GLP. Duplicate samples were taken for analysis at specified intervals up to 59 days after application. Traps for organic volatiles and carbon dioxide were used.The radioactivity was quantified by liquid scintillation counting and characterised by HPLC. LC-MS was used for confirmation of metabolites in selected samples.

The recovery in the water system was in the range of 91.0 % to 100.3 % applied radioactivity (AR) with the low test concentration and 88.2 % to 100.0 % AR with the high test concentration.

CO2 was observed in amounts up to 6.8 % and 5.2 % AR for the low and high test concentration, respectively. Organic volatiles were detected with mean amounts up to 14 % AR (low concentration) and 16.9 % AR (high concentration).

The compounds 2-tert-butyl-4-hydroxy-4,6-dimethylcyclohexa-2,5-dien-1-one (M5) and 3-tert-butyl-4-hydroxy-5-methylbenzoic acid (M6) were identified as major metabolites of the test item. M5 reached maximum values of 53.8 % AR (59 DAT) and 42.5 % AR (59 DAT) for the low and high test concentration, respectively. M6 was observed in amounts up to 16.7 % AR (low concentration) and 17.1 % AR (high concentration) at 59 DAT. Seven unknown peaks (M1, M2, M3, M4, M7, M8 and M12) in amounts <5 % AR (except M8 with 5.2 % AR at 29 DAT) were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M8, M9, M10, M11 and M13) were detected in amounts <5 % AR.

The test system was validated with the reference substance sodium benzoate. After 10 days 82.5 % AR of the reference item was mineralized. Thus, the system was biologically active.

The DT50 values for the test item were 11.5 days (9.5 μg/L) and 18.3 days (92.9 μg/L).

Reference 3

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with column 2 of REACH Annex IX, the study does not need to be conducted since the chemical safety assessment indicates that there is no need to investigate further the degradation of the substance and its degradation products.

Description of key information

Surface water. Key study. Experimental result: Test according to OECD guideline 309. GLP study. In a simulation biodegradation test performed in aerobic natural surface water, degradation half-time DT50 values for 2,5 -Dimethyl-4-tertbutyl[U-14C]phenol were 24.6 days (9.7 μg/L) and 117 days (91.3 μg/L).

Surface water. Key study. Experimental result: Test according to OECD guideline 309. GLP study. In a simulation biodegradation test performed in aerobic natural surface water, degradation half-time DT50 values for 2,4-Dimethyl-6-tertbutyl[U-14C]phenol were 11.5 days (9.5 μg/L) and 18.3 days (92.9 μg/L).

Surface water. Determination of key value for CSA: According to the chemical safety report, the estimated local PEC fresh water for the whole substance (multi-constituent substance) is 3.76E-6 mg/L. Therefore, the lower tested concentration in each test is the environmentally relevant one. Furthermore, the highest DT50 at the lowest concentration of the two tests has beeen finally selected as the worst case scenario.

Sediment. Data waiving: In accordance with column 2 of REACH Annex IX, the study does not need to be conducted since the chemical safety assessment indicates that there is no need to investigate further the degradation of the substance and its degradation products.

Key value for chemical safety assessment

Half-life in freshwater:

24.6 d

at the temperature of:

11.7 °C

Additional information

Surface water. Key study: A simulation biodegradation test was conducted for radiolabelled test item 2,5-Dimethyl-4-tertbutyl[U-14C]phenol with two different application rates (9.7μg/L and 91.3μg/L) under aerobic conditions in the dark using natural aerobic surface water from the large water body Rhineland-Palatinate (67374 Hanhofen, Germany). The study was conducted in accordance with the OECD Guideline 309 and GLP. Duplicate samples were taken for analysis at specified intervals up to 59 days after application.Traps for organic volatiles and carbon dioxide were used.The radioactivity was quantified by liquid scintillation counting and characterised byHPLC. LC-MS was used for confirmation of metabolites in selected samples.

The recovery in the water system was in the range of 86.8 % to 100.0 % applied radioactivity (AR) with the low test concentration and 87.5 % to 100.0 % AR with the high test concentration.

CO2 was observed in amounts up to 7.9 % and 2.4 % AR for the low and high test concentration, respectively. Organic volatiles were detected in maximal amounts of 3.5 % AR (low concentration) and 3.7 % AR (high concentration).

Compounds (4-tert-butyl-2,5-dimethylphenyl) sulfonic acid (M1), 5-tert-butyl-2-hydroxy-4-methylbenzoic acid (M4), 4-tert-butyl-5-(hydroxymethyl)-2-methylphenyl hydrogen sulfate (M4), 4-tert-butyl-2-formyl-5-methylphenyl hydrogen sulfate (M6) and M2 were identified as major metabolites of the test item. M1 reached maximum values of 36.1 % AR (45 days after treatment, DAT) and 16.6 % AR (59 DAT) for the low and high test concentration, respectively. M4 was observed in amounts up to 19.6% AR (low concentration) and 1.7% AR (high concentration) at 59 and 21 DAT, respectively. M6 was only detected in the samples of the low concentration in maximum amounts of 7.0 % AR after 59 DAT. M2 was only observed in the high concentrated samples in a maximum value of 9.6 % AR after 59 DAT. This metabolite could not be identified by LC-MS.

Six unknown peaks (M2, M3, M7, M8, M9 and M14) in amounts <5 % AR were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M5, M10, M11, M12 and M13) were detected in amounts <5 % AR.

The test system was validated with the reference substance sodium benzoate. After 10 days 82.5 % AR of the reference item was mineralized. Thus, the system was biologically active.

The DT50 values for the test item were 24.6 days (9.7μg/L) and 117 days (91.3μg/L).

Surface water. Key study: A simulation biodegradation test was conducted for radiolabelled test item 2,4-Dimethyl-6-tertbutyl[U-14C]phenol with two different application rates (9.5 μg/L and 92.9 μg/L) under aerobic conditions in the dark using natural aerobic surface water from the large water body Rhineland-Palatinate (67374 Hanhofen, Germany). The study was conducted in accordance with the OECD Guideline 309 and GLP. Duplicate samples were taken for analysis at specified intervals up to 59 days after application. Traps for organic volatiles and carbon dioxide were used.The radioactivity was quantified by liquid scintillation counting and characterised by HPLC. LC-MS was used for confirmation of metabolites in selected samples.

The recovery in the water system was in the range of 91.0 % to 100.3 % applied radioactivity (AR) with the low test concentration and 88.2 % to 100.0 % AR with the high test concentration.

CO2 was observed in amounts up to 6.8 % and 5.2 % AR for the low and high test concentration, respectively. Organic volatiles were detected with mean amounts up to 14 % AR (low concentration) and 16.9 % AR (high concentration).

The compounds 2-tert-butyl-4-hydroxy-4,6-dimethylcyclohexa-2,5-dien-1-one (M5) and 3-tert-butyl-4-hydroxy-5-methylbenzoic acid (M6) were identified as major metabolites of the test item. M5 reached maximum values of 53.8 % AR (59 DAT) and 42.5 % AR (59 DAT) for the low and high test concentration, respectively. M6 was observed in amounts up to 16.7 % AR (low concentration) and 17.1 % AR (high concentration) at 59 DAT. Seven unknown peaks (M1, M2, M3, M4, M7, M8 and M12) in amounts <5 % AR (except M8 with 5.2 % AR at 29 DAT) were observed in the test vessels with the low test concentration. For the high test concentrations five unknown peaks (M8, M9, M10, M11 and M13) were detected in amounts <5 % AR.

The test system was validated with the reference substance sodium benzoate. After 10 days 82.5 % AR of the reference item was mineralized. Thus, the system was biologically active.

The DT50 values for the test item were 11.5 days (9.5 μg/L) and 18.3 days (92.9 μg/L).

Sediment. Data waiving: In accordance with column 2 of REACH Annex IX, the study does not need to be conducted since the chemical safety assessment indicates that there is no need to investigate further the degradation of the substance and its degradation products.