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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Guideline:
other:
Version / remarks:
REACH guidance on QSARs R.6
Specific details on test material used for the study:
SMILES codes for calculation: see report T403_QSAR_24072018.docx in attached justification
Oxygen conditions:
aerobic
Inoculum or test system:
other: model calculation
Key result
Remarks on result:
other: the vast amount of potential metabolites of Jeffamine T403 predicted by the CRAFT model are predicted to be not readily biodegradable by the EPI Suite model
Validity criteria fulfilled:
yes
Conclusions:
Metabolites representing crucial steps in the transformation pathways (key metabolites) of Jeffamine T403 were identified by means of QSARs.
The CRAFT model identified an extensive amount of potential metabolites for Jeffamine T403. The biodegradability of each of these metabolites were predicted using the EPI Suite model. The vast majority of the metabolites were predicted to be not readily biodegradable.
Executive summary:

Given the final decision of ECHA (received on 19 July 2016) a simulation biodegradation study (EU C.25. / OECD 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test) typically carried out with 14C-labelled test substance must be performed. A QSAR exercise was completed to bolster the experimental testing and to guarantee meaningful endpoint coverage. Given the complex, partly unknown and variable composition of the parent compound Jeffamine T403 (CAS 39423-51-3), accurate assessment and experimental testing of all individual UVCB constituents is challenging. A QSAR exercise was performed aiming to identify the potential pathway each “representative” component of UVCB Jeffamine T403 could follow, under different combinations of environmental conditions (e.g. biotic, oxygen and compartments), explained in the document along with the likelihood at which the potential metabolites are formed. Once these potential pathways were constructed, the intermediates and end products were assessed for their P (B and T) properties in view of PBT/vPvB. For this supporting QSAR exercise, freely available tools and software such as CRAFT, EPI Suite models and packages within R statistical programming were used.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-18 - 2018-06-25 (pre-test); 2018-06-28 - 2018-09-25 (main test)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
yes
Remarks:
higher concentrations of the test item (i.e., > 100 µg/L) have been used to ensure successful quantification of the components of the UVCB monitored during the test (low analytical sensitivity)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: LL170303
- Expiration date of the lot/batch: 31 March 2019
- Purity test date: not applicable (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark over nitrogen

OTHER SPECIFICS:
- Molecular formula: not applicable (UVCB)
- Molecular weight: not applicable (UVCB)
- CAS-Number: 39423-51-3
- EC-Number: 500-105-6
- Water solubility (20°C, pH 12.7): 562 g/L
- Vapour pressure (20 °C): 682 Pa
- Density (20 °C): 0.966
Radiolabelling:
no
Remarks:
The study was accomplished with unlabelled test substance, the radiolabeling being unfeasible due to its nature (UVCB substance). The consideration of metabolites and the establishment of a mass balance were thus not possible.
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure):
*Name: Meschede Hennesee lake water
*Sampling location: Hennesee lake, 57392 Schmallenberg, North-Rhine-Westphalia, Germany
*Site description: A freshwater lake fed by a stream from a weir on the river. Woodland around the lake.
*Geographical region/global co-ordinates: Central Europe / 8°15’ East and 51°17’ Nord
*Date of collection: 14 June 2018, 11:15 a.m.
*Sampling depth: 0 – 40 cm
*Collection procedure: Immersion of container
- Storage conditions: samples kept waterlogged at ca. 4 ºC in the dark
- Duration of storage (prior to use): The test water was stored 14 days before preparing the test water samples.
- Temperature at collection (°C): 19.8
- pH at collection 7.647
- Oxygen concentration at collection (mg/L): 92.5%
- Optical appearance (NTU): No turbidity, colourless (0.48)
- Total Organic Carbon (mg/L): 22.56
- Dissolved Organic Carbon (mg/L): 2.17
- Biological Oxygen Demand (mg O2/L): < 0.1
- Nitrogen (total, mg/L): < 0.5
- Nitrate (mg/L): 2.5
- Nitrite (mg/L): 0.24
- Ammonium-N (mg/L): 0.3
- Total P (µg/L): 23.5
- Dissolved orthophosphate (total, mg/L): < 0.5
- Water filtered: yes
- Type and size of filter used, if any: Filtered through 100 µm mesh directly after sampling.
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): not determined
Details on source and properties of sediment:
not applicable
Details on inoculum:
not applicable
Duration of test (contact time):
60 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
PRE-TEST
In order to estimate appropriate sampling time points, to check whether there is adsorption to glassware and to check sample handling steps, a preliminary test with an incubation duration of 7 days was performed.

TEST CONDITIONS IN THE MAIN TEST
- Volume of test solution/treatment: 250 mL (into 0.5-L Erlenmeyer flasks)
- Preparation of media containing test item: Test item was added 4 days after preparing the test water samples. The required amount of Jeffamine T403 was solved in 10 mL distilled water and treated ultra-sonically for ca. 5 min to ensure homogeneity. The required volume of a test item solution with suitable concentration was applied by direct pipetting on the water surface and carefully shaken. Test concentrations were 2 and 14 mg of test item/L.
- Composition of medium: untreated natural river water
- Solubilising agent (type and concentration if used): none
- Test temperature: 20 ± 2°C
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: no

TEST SYSTEM
- Culturing apparatus: 0.5-L glass Erlenmeyer flasks (filling volume 250 mL), incubated on an orbital shaker (100 rpm)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions:
*Test item solutions: shaking + allowing gas exchange with the environment
*Biological activity control solutions: shaking + flow-through system in which a constant stream of surrounding air was passed over the control solutions
- Test performed in closed vessels due to significant volatility of test substance:
*Test item solutions: No. Non-sterile test samples were loosely covered by aluminum foil to allow gas exchange with the environment. Sterile solutions were covered with a sterile filter.
*Biological activity control solutions: Yes.
- Details of trap for CO2: Only relevant for the biological activity control samples. Outgoing gas was bubbled through two 14CO2 absorption traps in sequence containing 100 mL 1M NaOH each in order to determine the rate of mineralisation. Every sampling day the traps were replace by new onces until a mineralisation of 60% was reached.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: not applicable
- Abiotic sterile control: The test medium, test vessels, and all required equipment were autoclaved at 121°C for 15 min and the addition of the test item and homogenisation of the test solutions were performed under sterile conditions under a laminar flow workbench. The test item samples were filtered through a sterile filter before addition to the test medium. The test solutions were covered with a sterile filter.
- Toxicity control: not performed
- Biological activity control: The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls). The 14C-sodium benzoate stock solution (specific radioactivity 130 mCi/mmol) was diluted appropriately to result in the required treatment solution in distilled water. The required volume of this solution (96.14 µL equivalent to 83.45 kBq) was applied to the test solutions. The concentration of 14C-sodium benzoate was 10 µg/L.

SAMPLING
- Sampling frequency (test solutions and - for the biological activity control - 14CO2 absorption traps):
* Pre-test: days 0, 1, 3 and 7 after application
* Non-sterile test item solutions in main test: days 0, 7, 14, 22, 31, 45 and 60 after application
* Sterile test item solutions in main test: days 31 and 60 after application
* Biological activity control solutions in main test: days 1, 3, 7 and 14 after application
- Samples were taken from the orbital shaker (test item solutions) and - for the biological activity control - from the exchanged 14CO2 absorption traps.
- Sampling volume, dilution, and acidification (test solutions):
* An aliquot of 35 µL from the 14 mg/L test solution was taken and diluted to a final volume of 1 mL with blank test medium.
* An aliquot of 250 µL from the 2 mg/L test solution was taken and diluted to a final volume of 1 mL with blank test medium.
* To every diluted sample a volume of 10 µL formic acid was added.
- Sampling of 14CO2 absorption traps (biological activity control only):
* At every sampling occasion the 14CO2 absorption flasks were exchanged, the volume was determined and radioactivity measured.
- Sample storage before analysis: After sampling, the water samples were analysed immediately or stored at -20°C before analysis if required. An aliquot of 100 µL of every test solution was stored at -20°C at every sampling occasion for potential re-measurement. All measurements and re-measurements were performed within 35 days after sampling.
- Storage stability check: The 0-day samples were re-analysed (second analysis) 71 days after quantification (first analysis) and the results of first and second analysis compared. No significant deviation from the original measurement was detected. For component 1 to 6, 92.2-105.7% of the original amount was found after re-measurement. The difference was slightly higher for component 7 (108.2 and 113.9%).
Reference substance:
benzoic acid, sodium salt
Test performance:
The performance of the test system was evaluated using the 14C-labelled reference substance sodium benzoate. After 7 days of incubation > 60% of the applied radioactivity was mineralised in both control samples. Therefore, the test water was considered sufficiently biologically active.
Compartment:
natural water: freshwater
% Recovery:
104
Remarks on result:
other: Average recovery at t=0h in the 2 mg/L test solution (excluding the results for component 7)
Remarks:
Range: 98.3-108.1%
Compartment:
natural water: freshwater
% Recovery:
117.6
Remarks on result:
other: Average recovery at t=0h in the 14 mg/L test solution (excluding the results for component 7)
Remarks:
Range: 112.0-125.3%
Compartment:
biologically active treatment at end of test
% Recovery:
90.2
Remarks on result:
other: Average (corrected) recovery at t=60d in the 2 mg/L test solution (excluding component 7, for which results were not valid)
Remarks:
Range: 79.6-112.5% (or, without the single test solution with recovery 112.5%: 79.6-98.0%)
Compartment:
biologically active treatment at end of test
% Recovery:
94.9
Remarks on result:
other: Average (corrected) recovery at t=60d in the 14 mg/L test solution (excluding component 7, for which results were not valid)
Remarks:
Range: 85.8-113.2% (or, without the single test solution with recovery 113.2%: 85.8-100.5%)
Compartment:
abiotic control measured at end of test
% Recovery:
100.4
Remarks on result:
other: Average (corrected) recovery at t=60d in the 2 mg/L test solution (excluding component 7, for which results were not valid)
Remarks:
Range: 90.2-132.3%
Compartment:
abiotic control measured at end of test
% Recovery:
98.4
Remarks on result:
other: Average (corrected) recovery at t=60d in the 14 mg/L test solution (excluding component 7, for which results were not valid)
Remarks:
Range: 88.4-124.3%
Key result
% Degr.:
9.78
Parameter:
test mat. analysis
Remarks:
7 components of the UVCB were monitored
Sampling time:
60 d
Remarks on result:
other: On average, in the 2 mg/L test solution, 9.78% degradation was observed after 60 days (range: -12.5-20.4%) based on analysis of 6 components of the UVCB (results for component 7 omitted since not valid).
Key result
% Degr.:
5.11
Parameter:
test mat. analysis
Remarks:
7 components of the UVCB were monitored
Sampling time:
60 d
Remarks on result:
other: On average, in the 14 mg/L test solution, 5.11% degradation was observed after 60 days (range: -13.2-14.2%) based on analysis of 6 components of the UVCB (results for component 7 omitted since not valid).
Key result
Compartment:
natural water
DT50:
>= 179 - <= 372 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: range of DT50 values for the components monitored in the 2 mg test item/L test solution (component 6 excluded because the Prob. > t (k_deg) values were > 0.1; component 7 excluded since a number of HR LC-MS/MS quantifications were not valid)
Key result
Compartment:
natural water
DT50:
>= 298 - <= 464 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: range of DT50 values for the components monitored in the 14 mg test item/L test solution (component 6 excluded because the Prob. > t (k_deg) values were > 0.1; component 7 excluded since a number of HR LC-MS/MS quantifications were not valid)
Transformation products:
not measured
Remarks:
The study was accomplished with unlabelled test item because the synthesis of radiolabelled substance was not considered feasible. The identification and monitoring of metabolites was therefore not possible.
Evaporation of parent compound:
not measured
Remarks:
Evaporation was considered highly unlikely based on the low vapour pressure and Henry coefficient of the substance.
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
TEST CONDITIONS
- Temperature was 20°C +/- 2°C during the first 21 days. Due to a malfunction of the temperature logger, test temperature data from day 22 up to day 60 are not available. However, the climate controlling system was active over the complete incubation time and a min/max thermometer confirmed a temperature of > 17°C and < 23°C over the last few years. Therefore, there were either no or no more than negligible deviations from the required temperature range during the test.
- pH and oxygen concentrations were measured at days 0, 7, 14, 22, 31, 45 and 60 and ranged from 8.29 to 8.36 and from 7.23 to 8.27 mg/L, respectively.

VERIFICATION OF TEST ITEM CONCENTRATION AT t=0h
*2 mg/L test solution: Recovery ranging from 98.3 to 108.1%. Only 81.7% was found for component 7 in one replicate, however, component 7 was difficult to quantify by HR LC-MS/MS and the results at t=14, 22, 31 and 60 days were not always valid, therefore results for component 7 should be interpreted with care.
*14 mg/L test solution: Recovery ranging from 112.0 to 125.3%. The test item concentration was slightly higher than intended. Again, results for component 7 should be interpreted with care because the component was difficult to quantify by HR LC-MS/MS.

TEST ITEM CONCENTRATION DURING INCUBATION
*2 mg/L: The degradation in the test water is very slow. After 60 days of incubation the recovery ranged between 80 and 98% (in one replicate a recovery of 112.5% was found). The results for component 7 were not valid.
*14 mg/L: The degradation in the test water was very slow. After 60 days of incubation the recovery ranged between 86 and 100.5% (in one replicate a recovery of 113.2% was found). The results for component 7 were not valid.
*Degradation is observed in both sterile and non-sterile samples, hence abiotic degradation is also a relevant factor in the degradation behaviour of the components followed. However, a clearly higher degradation was mostly found in non-sterile samples. Therefore, both biotic and abiotic degradation are considered relevant.

DT50 CALCULATIONS
- Calculations were performed using CAKE version 1.4 (Computer Aided Kinetic Evaluation).
- For calculations, the recoveries obtained for the corresponding components 1 to 7 were used.
- Analyses were based on single first order, double first order in parallel, and Hockey stick kinetics. First order multi-compartment kinetics were not considered since the DT90 was never reached during the study. Based on the results of the analyses, there is no evidence for biphasic kinetics and therefore the results according to single first order kinetics were considered for further use.
- Ranges of the DT50 values for components 1 to 5 are given in the results table above.
- DT90 values: 594-1236 days (2 mg/L) and 990-1541 days (14 mg/L). As for the DT50 values, components 6 and 7 were not regarded for the same reasons as given in the table.
Results with reference substance:
After seven days of incubation 61.3-64.4% of the 14C-sodium benzoate was already mineralised. After 14 days (end of incubation) mineralisation was 70.0-70.3%.

Table 1. Verification of test item concentration for the 7 components separately at t=0h in the 2 mg test item/L test solution.

Test component

Test replicate

Recovery [%]

1

A

103.5

B

105.8

2

A

105.6

B

101.7

3

A

103.4

B

103.0

4

A

104.7

B

103.4

5

A

108.1

B

105.8

6

A

104.4

B

 98.3

7

A

101.5

B

 81.7

 

Table 2. Verification of test item concentration for the 7 components separately at t=0h in the 14 mg test item/L test solution.

Test component

Test replicate

Recovery [%]

1

A

112.7

B

117.9

2

A

116.7

B

117.9

3

A

112.0

B

116.3

4

A

115.8

B

119.2

5

A

120.0

B

122.6

6

A

114.4

B

125.3

7

A

106.7

B

120.6

Table 3. % recovery for the 7 components over time in the 2 mg test item/L test solution.

Test component

Test replicate

Recovery [%]

 

 

0 days

7 days

14 days

22 days

31 days

45 days

60 days

1

A

100.0

104.7

100.8

94.6

93.3

86.5

79.6

B

100.0

105.7

99.5

89.3

93.7

84.6

85.6

2

A

100.0

99.7

98.7

93.3

92.5

88.3

80.5

B

100.0

110.4

106.7

92.1

96.6

92.3

92.7

3

A

100.0

99.0

103.1

98.1

96.1

92.5

86.4

B

100.0

106.9

106.9

93.5

98.3

94.5

92.6

4

A

100.0

101.5

107.4

98.9

95.2

93.8

85.1

B

100.0

107.3

111.4

93.5

98.4

95.2

91.1

5

A

100.0

95.3

108.9

97.3

94.9

92.0

88.2

B

100.0

107.5

113.8

93.1

96.8

97.4

98.0

6

A

100.0

89.9

115.6

99.8

99.1

84.6

90.3

B

100.0

113.6

120.3

98.5

102.2

81.4

112.5

7

A

100.0

78.6

115.61

102.71

101.1

94.01

87.91

B

100.0

120.8

147.21

114.31

93.0

99.91

141.01

1LC-MS/MS data not valid, quality control sample deviation >15% for the respective component

Table 4. % recovery for the 7 components over time in the 14 mg test item/L test solution.

Test component

Test replicate

Recovery [%]

 

 

0 days

7 days

14 days

22 days

31 days

45 days

60 days

1

A

100.0

107.3

96.7

92.4

96.8

93.6

99.4

B

100.0

102.5

91.8

94.0

87.2

93.4

86.8

2

A

100.0

102.7

91.0

89.0

91.0

89.0

93.2

B

100.0

102.7

95.2

92.1

87.3

88.6

85.8

3

A

100.0

104.0

96.6

94.1

96.3

94.5

96.7

B

100.0

100.2

97.8

94.8

87.9

90.7

88.2

4

A

100.0

100.5

99.7

92.8

94.6

91.0

94.7

B

100.0

99.3

99.4

95.4

88.6

90.2

86.6

5

A

100.0

103.3

100.7

93.3

96.6

89.0

98.9

B

100.0

100.5

104.1

99.3

87.3

92.2

94.7

6

A

100.0

117.0

106.9

97.7

103.4

92.9

113.2

B

100.0

95.2

101.8

101.6

84.6

96.7

100.5

7

A

100.0

139.4

107.81

110.81

129.1

103.01

123.41

B

100.0

95.1

114.91

119.41

92.4

190.51

107.21

1LC-MS/MS data not valid, quality control sample deviation >15% for the respective component

Table 5. Comparison of % recovery for the 7 components after 31 and 60 days in non-sterile versus sterile samples in the 2 mg test item/L test solutions.

Test component

Test replicate

Recovery [%]

 

 

31 days

non-sterile

31 days

sterile

60 days

non-sterile

60 days

sterile

1

A

93.3

98.8

79.6

92.8

B

93.7

92.4

85.6

90.2

2

A

92.5

94.8

80.5

91.5

B

96.6

96.1

92.7

92.2

3

A

96.1

98.6

86.4

94.7

B

98.3

94.7

92.6

94.5

4

A

95.2

100.0

85.1

94.2

B

98.4

94.4

91.1

99.5

5

A

94.9

99.3

88.2

100.2

B

96.8

95.4

98.0

108.3

6

A

99.1

109.0

90.3

114.9

B

102.2

102.5

112.5

132.3

7

A

101.1

132.2

87.91

122.21

B

93.0

124.1

141.01

172.31

1LC-MS/MS data not valid, quality control sample deviation >15% for the respective component

Table 6. Comparison of % recovery for the 7 components after 31 and 60 days in non-sterile versus sterile samples in the 14 mg test item/L test solutions.

Test component

Test replicate

Recovery [%]

 

 

31 days

non-sterile

31 days

sterile

60 days

non-sterile

60 days

sterile

1

A

96.8

102.1

99.4

95.3

B

87.2

103.1

86.8

92.0

2

A

91.0

95.6

93.2

88.4

B

87.3

95.8

85.8

89.6

3

A

96.3

100.2

96.7

95.6

B

87.9

98.5

88.2

92.5

4

A

94.6

98.7

94.7

97.5

B

86.6

98.1

86.6

94.5

5

A

96.6

99.4

98.9

102.6

B

87.3

99.3

94.7

100.7

6

A

103.4

105.6

113.2

124.3

B

84.6

97.9

100.5

108.2

7

A

129.1

122.3

123.41

146.61

B

92.4

113.5

107.21

 112.51

1LC-MS/MS data not valid, quality control sample deviation >15% for the respective component

Table 7. Mineralisation of 14C-sodium benzoate in the biological activity control samples.

Control sample replicate

Incubation time

Mineralised as14CO2

A

0 days

0,0%

B

0,0%

A

1 day

 

25,8%

B

28,5%

A

3 days

 

50,2%

B

49,3%

A

7 days

 

64,4%

B

61,3%

A

14 days

 

70,0%

B

70,3%

Table 8. CAKE calculations for DT50 and DT90 of the 7 monitored components of the UVCB based on single first order kinetics.

Concentration

Peak

SFO

 

 

chi²

(%)

(-)

Prob. > t (k_deg)

 

DT50

(d)

DT90

(d)

Low

1

2.279

0.8368

2.863E-06

178.8

594

 

2

2.329

0.6027

0.0006171

231.4

768.6

 

3

2.05

0.6195

0.0004688

307.9

1023

 

4

3.133

0.5693

0.001047

264

877.1

 

5

3.652

0.2904

0.02439

372

1236

 

62

6.807

0.1092

0.1259

366.4

1217

 

71,2

8.685

0.0001748

0.5

1.028 10+12

3.415 10+12

High

1

2.714

0.2925

0.02276

434.5

1443

 

2

2.682

0.5692

0.0009091

298.1

990.3

 

3

1.618

0.499

0.002394

411.3

1366

 

4

1.587

0.6306

0.00036

335.1

1113

 

5

2.658

0.3242

0.01668

463.8

1541

 

62

3.779

0.003151

0.4235

3122

1.037 10+04

 

71,2

9.106

0.08722

0.5

4.462 10+14

1.482 10+15

 

1A lot of HR LC-MS quantifications are not valid and therefore the result should be considered critically

2Based on the Prob. > t (k_deg) values (>0.1) the DT50 results are not valid.

Validity criteria fulfilled:
yes
Conclusions:
The biodegradation of the test substance was studied in an OECD 309 study using natural water at 2 concentrations of the test item (i.e., 2 and 14 mg/L). The test item concentrations were higher than recommended by the guideline to ensure succesful analytical quantification, because the analytical sensitivity (HR LC-MS/MS) of the 7 components of the UVCB monitored during the test was rather low.

First, a 7-day preliminary test was performed in order to estimate appropriate sampling time points, to check whether there is adsorption to glassware and to check sample handling steps. After 7 days of incubation, no clear disappearance of the main components was observed. No indication of adsorption to glassware was observed.

The duration of the main study was 60 days. On average, 9.78 and 5.11% degradation was observed (average calculated excluding component 7 because not all results were valid for this component) by the end of the study in the 2 mg/L and 14 mg/L test item solution. A comparison of the results obtained in biologically active test solutions with those obtained in sterile test solutions learned that additionally, abiotic degradation may contribute (be it to a lesser extent) to the total observed degradation of the components. The results obtained with the reference substance 14C-sodium benzoate indicated that the natural water was sufficiently biologically active and therefore suitable for the test. Since radiolabelling of the test item was not possible due to the nature of the test material (UVCB), the monitoring of transformation products and the establishment of a mass balance were not possible. Based on the recoveries measured over time for the different components, DT50 values according to single first order kinetics were calculated using the CAKE model (Computer Aided Kinetic Evaluation). The DT50 values based on the recoveries measured in the 2 mg/L test solution ranged from 179 to 372 days, whereas those based on the recoveries mentioned in the 14 mg/L test solution ranged from 298 to 464 days. These DT50 ranges do not include the DT50 values obtained for components 6 and 7 because these were not considered sufficiently reliable.

In conclusion, based on the results of this study, the (bio)degradation of the test item in natural test water is very slow.
Endpoint:
biodegradation in water: sediment simulation testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Description of key information

One experimental study is allocated to this endpoint: an aerobic mineralisation study in surface water, performed according to OECD guideline 309 (Gärtner, 2019). The study is considered as key study and reliable without restrictions (Klimisch 1). Limited (bio)degradation was observed in this study, resulting in DT50 values between

179 and 464 days depending on the component of the UVCB followed during the study as well as on the initial concentration of the test item.

Key value for chemical safety assessment

Half-life in freshwater:
179 d
at the temperature of:
20 °C

Additional information

Data on the degradation behaviour of the test substance (CAS 39423-51-3) in natural water is required in order to further assess the fate of the substance in the aquatic environment. A study was therefore carried out according to OECD guideline 309 (Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test) in accordance with the principles of Good Laboratory Practice (Gärtner, 2019).

This study was concerned with the disappearance of seven representative components of the UVCB in natural surface water under well defined and controlled laboratory conditions in in order to evaluate the potential for microbial and chemical degradation. The study was accomplished with unlabelled test substance, the radiolabelling being unfeasible due to the nature of the substance (UVCB). Consequently, monitoring of transformation products and the establishment of a mass balance were not possible. Further, due to technical limitations (low analytical sensitivity), the test item concentrations were higher (i.e., 2 and 14 mg test item/L) than recommended by the guideline (i.e. <= 100 µg/L), to guarantee successful quantification of all components that were intended to be monitored. The study started with a 7-day preliminary test, during which no clear disappearance of the seven representative components of the test substance was observed.

In the main study, which was performed over a 60-day period, 9.78% degradation was observed on average in the 2 mg/L test solutions, whereas in the 14 mg/L test solutions average degradation was 5.11%. Estimated DT50 values for the 2 mg/L test solutions ranged from 179 to 372 days depending on the component under consideration. In the 14 mg/L test solutions the estimated DT50 values ranged from 298 to 464 days. DT90 values were estimated to range from 594 to 1236 days (2 mg/L) and from 990 to 1541 days (14 mg/L), depending on the component. DT50 and DT90 estimates for component 6 and 7 were not considered as they were not considered to be sufficiently reliable. Further, a comparison of the recovery of the 7 components over time in biologically active test solutions with that observed in abiotic test solutions with the same loading learned that a limited amount of degradation was generally also observed in abiotic test solutions, indicating a limited contribution of abiotic degradation to the observed total degradation. Based on these results, degradation of the test substance in natural water is concluded to be very slow (i.e. the substance is not rapidly degradable). Although abiotic degradation was also observed to a limited extent, biodegradation is considered relatively more important than abiotic degradation in the removal of the test substance from natural water through degradation processes.

Given the complex, partly unknown and variable composition of the parent compound, accurate assessment of the degradation behaviour of all individual UVCB constituents is challenging. A QSAR exercise was therefore performed aiming to identify the potential pathway each “representative” component of the test substance could follow. Once these potential pathways were constructed using the CRAFT model, the intermediates and end products were assessed for their P, (B and T) properties in view of the PBT/vPvB screening assessment. The CRAFT model identified an extensive amount of degradation products for the test substance. The biodegradability of each of these degradation products was predicted using the EPI Suite model. The vast majority of the degradation products was predicted not to be readily biodegradable.