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Biodegradation in water and sediment: simulation tests

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Reference
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Remarks:
Purpose of the study was to investigate aerobic transformation rate and route of [phenyl-U-¹⁴C]2,4,6-trimethylphenol in natural water at low conc.. The degradation rate of the test item, mass balance and transformation product pattern were determined.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 14, 2018 - November 25, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was based on OECD Guideline for Testing of Chemicals No. 309: "Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test”; adopted April 13, 2004.
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
yes
Remarks:
Test performed under GLP according to guideline with some minor acceptable deviations (recovery were partly belowe the target value). Test meets all validity criteria.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The synthesis of [phenyl-U-14C]2,4,6-trimethylphenol was performed by
Selcia Limited - Fyfield Business & Research Park, Fyfield Road, Ongar, Essex, CM5 0GS, United Kingdom.
It was necessary to synthetize two batches.

1st and 2nd Approach: it was used Batch 5736JYC027-2
Specific Activity: 2749 MBq/mmol (74.29 mCi/mmol), 19.84 MBq/mg (536.1 μCi/mg)
Molecular Weight:138.57 g/mol @ 74.29 mCi/mmol
Radiochemical Purity:99.0% according to the CoA
Chemical Purity:99.5% according to th CoA
Certificate of Analysis Date:November 20, 2017
Physical Appearance / Colour:Solid, white
Expiry Date: not applicable. The amount of total radioactivity was determined by LSC and absence of degradation products was verified by HPLC.
Storage: At ≤ -50°C, in the dark

3rd Approach: it was used Batch 10191KEP018-3
Specific Activity: 2679 MBq/mmol (72.41 mCi/mmol), 19.34 MBq/mg (522.8 μCi/mg)
Molecular Weight: 138.51 g/mol
Radiochemical Purity: 98.3 % according to CoA
Chemical Purity: 98.4 % according to CoA
Certificate of Analysis Date: May 16, 2019
Physical Appearance / Colour: Solid, pale yellow
Expiry Date: Not applicable. The amount of total radioactivity was determined by LSC and absence of degradation products was verified by HPLC.
Storage: At ≤ -50°C, in the dark
Radiolabelling:
yes
Oxygen conditions:
aerobic
Remarks:
The oxygen saturation of the water was ≥ 80%
Inoculum or test system:
natural water: freshwater
Details on source and properties of surface water:
Two types of surface water were used for the tests because the dry weather conditions with low amounts of rain led to a missing inflow of fresh water into the sampling site (pond) which resulted in unnatural brackish water.
• 1st and 2nd approach: pond.
Details on collection: Location Ludwigsteich, Rossdorf, Germany (coordinates 49.861766, 8.727451)
Date of sampling 03/2018 apperance yellowish, clear
- pH at time of collection: 8.1
- Redox potential (mv): 134
- Oxygen concentration (mg/l): 11.6
Date of sampling 09/2018 apperance yellowish/brownish, turbid
- pH at time of collection: 7.7
- Redox potential (mv): 137
- Oxygen concentration (mg/l): 5.2

• 3rd approach: river
Details on collection: River Rhein, Germany (Coordinates 49.774247, 8.418256)
Date of Sampling 07/2019 Appearance slightly yellow, clear
- pH at time of collection: 7.8
- Redox potential (mv): 149
- Oxygen concentration (mg/l): 7.9

The water was transported in glass bottles. During transport, the temperature of the sample did not exceed the temperature used in the test.
Prior to use, coarse particles were removed by filtration (100 μm).
Duration of test (contact time):
60 d
Initial conc.:
10 µg/L
Based on:
test mat.
Remarks:
(low)
Initial conc.:
100 µg/L
Based on:
test mat.
Remarks:
(high)
Initial conc.:
1 000 µg/L
Based on:
test mat.
Remarks:
(initially prepared for structure elucidation of unknown transformation products)
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Test Setup:
The test was conducted as multiple flasks approach.

Test Vessels:
Glass bottles (250 mL and 500 mL); continuous flow-through; stirring. Each flask was equipped with traps to absorb volatile degradation products (except for the sterile samples which were closed hermetically).

Absorption Material:
1st approach:
35 mL water
35 mL ethylene glycol
35 mL 0.05 M H2SO4
35 mL 2 M NaOH
35 mL 2 M NaOH
2nd approach:
20 mL water
20 mL ethylene glycol
20 mL 0.05 M H2SO4
20 mL 2 M NaOH
20 mL 2 M NaOH
3rd approach:
20 mL water
20 mL ethylene glycol
20 mL 0.05 M H2SO4
30 mL 2 M NaOH
30 mL 2 M NaOH

Test Conditions
Temperature: 20°C ± 2°C
Light Conditions: Darkness
Microbial Activity: Microbial activity of the surface water was verified by following the degradation of 14C-labelled benzoic acid in the control samples.
Aerobic Incubation Conditions: Aerobic incubation conditions were monitored by measuring the oxygen content and redox potential of the surface water

Stock Solution of the Test Item:
The following stock solutions were prepare in Acetonitrile :
A. 313 mg/L (6.2 MBq/mL) not used for application
B. 333 mg/L (6.6 MBq/mL)
C. 273 mg/L (5.3 MBq/mL)
The solutions were stored in the freezer at < - 20°C until further use.

Application Solution of the Test Item: Either the stock solution was used for application (samples of conc. II and III) or the stock solution was diluted prior to application (samples of conc. I).
For dilution Acetonitrile was used. The following diluted solutions were prepared:
D. 29.9 mg/L (0.6 MBq/mL)
E. 38.1 mg/L (0.8 MBq/mL)
F. 28.7 mg/L (0.6 MBq/mL)


Course of the Study
Water samples were incubated and samples were taken at specific time points. Extractable radioactivity, remaining radioactivity in the aqueous phase and volatile transformation products were investigated.
Mass balances and transformation product pattern were determined.
In terms of the 2nd approach the transformation product pattern could not be determined due to the water properties.

1st Approach

Test Duration: The incubation of treated samples was terminated after 60 days.
Sampling of Test Samples: 0, 8, 14, 21, 30, 44, 60 days (duplicate sampling)
Sampling of Sterile Samples: 0, 8, 14, 21, 30, 44, 60 days (single samples)
Sampling of Absorption Material: The traps were sampled together with the test samples. Samples taken immediately after application (0 days) were prepared without traps.

2nd Approach

Test Duration: The incubation of treated samples was aborted after 50 days due to the water properties.
Sampling of Test Samples: Conc I: 0, 7, 14, 50 days (duplicate sampling, except after 50 days)
Conc III: 14 days (duplicate sampling)
Sampling of Sterile Samples: 0, 7, 14 days (single samples)
Sampling of Absorption Material: The traps were sampled together with the test samples. Samples taken immediately after application (0 days) were prepared without traps.

3rd Approach

Test Duration: The incubation of treated samples was terminated after 60 days.
Sampling of Test Samples: 0, 1, 2, 4, 7, 14, 23, 35, 46, 60 days (duplicate sampling)
Sampling of Sterile Samples: 0, 1, 2, 4, 7, 14, 23, 35, 46, 60 days (single samples)
Sampling of Absorption Material: The traps were sampled together with the test samples. Samples taken immediately after application (0 days) were prepared without traps.

Determination of Total Radioactivity and Purity:
Total radioactivity in the test item stock and application solutions was determined by LSC. Purity of the labelled test item was verified by HPLC radio detection prior to application. Radiochemical purity was found to be in the range of 98-100%.
Reference substance:
other: [7-14C] Benzoic Acid
Reference substance:
other: 2,4,6-Trimethylphenol
Remarks:
Non-labelled 2,4,6-Trimethylphenol
Test performance:
The pelagic test system was chosen for this study using two types of surface water. Water samples were incubated and samples were taken at specific time points. Extractable radioactivity and volatile transformation products were investigated. The test item and the unknown transformation products in the extracts were analysed by HPLC with radio detection and HR-LC-MS/MS.
Key result
% Degr.:
ca. 100
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Conc. 10 μg/L 1st approach
Remarks:
After 60 days a complete degradation of the test item was observed.
Key result
% Degr.:
ca. 100
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Conc. 100 μg/L 1st approach
Remarks:
After 60 days a complete degradation of the test item was observed.
Key result
% Degr.:
ca. 100
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Conc. 10 μg/L 3rd approach
Remarks:
After 60 days a complete degradation of the test item was observed.
Key result
% Degr.:
ca. 100
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Conc. 100 μg/L 3rd approach
Remarks:
After 60 days a complete degradation of the test item was observed.
Compartment:
natural water: freshwater
DT50:
2.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: conc. 10 μg/L 1st approach
Key result
Compartment:
natural water: freshwater
DT50:
10.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: conc. 100 μg/L 1st approach
Compartment:
natural water: freshwater
DT50:
2 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: conc. 10 μg/L 3rd approach
Compartment:
natural water: freshwater
DT50:
3.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: conc. 100 μg/L 3rd approach
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
Details on results:
Test Conditions
Temperature: 20°C ± 2°C
Light Conditions: Darkness
Microbial Activity: Microbial activity of the surface water was verified by following the degradation of 14C-labelled benzoic acid in the control samples.
Aerobic Incubation Conditions:
Aerobic incubation conditions were monitored by measuring the oxygen content and redox potential of the surface water
MAJOR TRANSFORMATION PRODUCTS
After 60 days, the test item amount was For the three major products HR-LC-MS/MS was performed to identify the structure. Hereto selected samples of the first and third approach were analysed exemplarily:
- 1st approach: conc. I: 14 d, 60 d; conc. II: 21 d, 60 d
- 3rd approach: conc. I: 14 d, 60 d; conc. II: 14 d, 60 d
Structure proposals were made based on the exact masses and fragmentation pattern.
1st approach: The three major transformation products were assigned to DHP, 6-HTDO/4-HTDO and DMS/HDMB. DHP was a highly degraded transformation product with a maximum amount of 5.2% AR (conc. I) after 44 days. 6-HTDO/4-HTDO were found with a maximum amount of 84.5% AR (conc. I) and 86.7% AR (conc. II) after 21 and 44 days, respectively. DMS/HDMB was observed with a maximum amount of 15.2% AR (conc. I) and 12.3% AR (conc. II) after 8 days of incubation.
3rd approach: The two major transformation products were assigned to DHP and 6-HTDO/4-HTDO. DHP was observed with a maximum amount of 25.6% AR (conc. I) and 17.4% AR after 60 and 23 days, respectively. 6-HTDO/4-HTDO were found with a maximum amount of 75.0% AR (conc. I) and 80.2% AR (conc. II) after 14 and 35 days, respectively.
Validity criteria fulfilled:
yes
Conclusions:
Degradation (after the lag phase) of the test item was extraordinary fast, the degradation after 60 days was complete (recoveries
Executive summary:

The purpose of this study was to investigate the aerobic transformation rate and route of [phenyl-U-¹⁴C]2,4,6-trimethylphenol in natural water at low test item concentrations. The degradation rate of the test item, mass balance and transformation product pattern were determined.

The pelagic test system was chosen for this study using two types of surface water: pond (1st and 2nd approach) and river (3rd approach). Based on the properties of the surface water the second approach was aborted during incubation.

This summary includes the results of the first and third approach. Two test item concentrations of 10 μg/L (conc. I) and 100 μg/L (conc. II) were used. The test vessels were attached to a flow-through system for continuous aeration of the surface water and incubated in the dark, samples were taken at specific time points.

Overall aerobic conditions during the test were verified for the test samples and the samples of the viability test. The oxygen saturation of the water was ≥ 80% and the redox potential ≥ +111 mV.

Sufficient microbial activity of the surface water was shown by a complete degradation (after 1-4 days of incubation) of the benzoic acid reference item (viability test). Validity of sample preparation was verified by calculating procedural recoveries.

Extractable radioactivity and volatile transformation products were investigated. For samples of conc. I recoveries were partly < 90% of the initial radioactivity indicated a loss of radioactivity during sample preparation which could be traced back to the transformation product properties. Regarding the loss of radioactivity during sample preparation, several tests were performed to improve the procedural recoveries prior to the third approach. Those tests were based on SPE extraction, freeze drying conditions and test item concentrations. Therecoveries were higher as compared to the first approach but still partly < 90% of the initial radioactivity especially for the samples of conc. I. Based on the efforts made and the improved results during the third approach, the results were considered acceptable to evaluate the behavior of the test item at low concentrations in natural surface water.

The test item and the unknown transformation products in the extracts were analyzed by HPLC with radio detection and HR-LC-MS/MS.

Structure proposals were made based on the exact masses and fragmentation pattern.

The signal at 1.6-1.7 min was assigned to: 2,2-dihydroxypropanoic acid (DHP) [CAS No. 1825-45-2]

For the signal at 5.6-6.4 min: A: 6-hydroxy-2,4,6-trimethylcyclohexa-2,4-dien-1-one (6-HTDO) [CAS No. 65810-46-0] // B: 4-hydroxy-2,4,6-trimethylcyclohexa-2,5-dien-1-one (4-HTDO) [CAS No. 16404-66-3] (With the analytical method used a differentiation between both structures was not feasible.)

For the signal at 6.9-7.0 min: A: 3,5-dimethylsalicylic acid (DMS) [CAS No. 6245-04-1] // B: 4-hydroxy-3,5-dimethylbenzoic acid (HDMB) [CAS No. 4919-37-3] (With the analytical method used a differentiation between both structures was not feasible.)

DT50 and DT90 (degradation time) values were calculated by using the test item specific results of HPLC radio detection analysis. If a lag phase was observed, values of the lag phase and the “tailing” were not considered for result evaluation. For the first approach, a lag phase was not observed as the sampling intervals (at the beginning) were not appropriate. During the third approach sampling intervals were adjusted at the beginning and hence the lag phase could be determined at both concentrations which lasted for approx. 7 days. In both cases, first and third approach, the degradation (after the lag phase) of the test item was extraordinary fast and thus only a few sampling points covering the degradation of the test item down to 50% AR or 10% AR. However, DT50 and DT90 values were calculated based on the available results (even if only a few sampling points were available).

 

 conc. I (10 μg/L) 1st approach

 conc. II (100 μg/L) 1st approach

 conc. I (10 μg/L) 3rd approach

 conc. II (100 μg/L) 3rd approach

 DT50

 2.7 d

10.8 d 

 2.0 d

 3.6 d

 DT90  9.0 d  35.7 d  6.6 d  11.9 d

Description of key information

Test on [phenyl-U-¹⁴C] 2,4,6-trimethylphenol: Aerobic Mineralisation in Surface Water – Simulation Biodegradation [OECD 309]

The purpose of this study was to investigate the aerobic transformation rate and route of [phenyl-U-¹⁴C]2,4,6-trimethylphenol in natural water at low test item concentrations. The degradation rate of the test item, mass balance and transformation product pattern were determined.

Performance of the Test: The pelagic test system was chosen for this study using two types of surface water. Water samples were incubated and samples were taken at specific time points. Extractable radioactivity and volatile transformation products were investigated. The test item and the unknown transformation products in the extracts were analysed by HPLC with radio detection and HR-LC-MS/MS.

Test Conditions: 20°C ± 2°C, in the dark

Treatment Rate:

Test item concentration I: 10 μg/L (low)

Test item concentration II: 100 μg/L (high)

Key value for chemical safety assessment

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

Additional information