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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2019 - 22 August 2019
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)
Version / remarks:
April 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Non-radiolabelled test item:
- Source and lot/batch No.of test material: 1902261800R
- Expiration date of the lot/batch: 26 February 2021
- Purity: 99.95%
- Storage: +20 ± 5°C in the dark

RADIOLABELLING INFORMATION (if applicable)
Radiolabelled test item:
- Specific activity: 12.7 MBq/mg
- Radiochemical purity: > 99%
- Locations of the label: * [methane-14C]
- Source and lot/batch No.of test material: 190221
- Storage: -20 ± 10°C
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: surface water
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Surface water was provided by Covance as part of this study.
Preliminary experiments 1 and 3: water was transported in containers and, upon receipt at the testing facility and prior to use, the surface water was passed through a 0.2 mm sieve and a coarse filter paper (GF/B).
Preliminary experiment 2 only, water was transported in containers and, upon receipt at the testing facility and prior to use, the surface water was passed through a coarse filter paper (GF/A).
The surface water was either used on the day of collection or stored at 2 - 8°C until required.
Characterization of the surface water was carried out, not to GLP, by Natural Resource Management Laboratories Ltd. (Berkshire, UK).

Physical and Chemical Characteristics of the Surface Water (Batch H090819A) used for Preliminary Experiment 3:
Physical/chemical characteristic: Result
Total organic carbon (mg/L): 6.7
Dissolved organic carbon (mg/L): 6.7
Biological oxygen demand (mg/L): <2
Total nitrogen (mg/L): 9.6
Total phosphorus (mg/L): 0.4
Ammonium nitrogen (mg/L): 2.8
Nitrate nitrogen (mg/L): 6.6
Nitrite nitrogen (mg/L): <0.1
Soluble reactive phosphorus (μg/L): 378
Total suspended solids (mg/L): <10
Hardness as CaCO3 (mg/L): 353

Surface Water Sampling Information
Preliminary Experiment 1
Origin: Great Ouse River, Huntingdon, Cambridgeshire, UK
Date of sampling: 07 March 2019
Water body type: River
Appearance: Clear, yellow/green in colour
Sampling depth (cm): 130
Temperature (°C), just below water surface: 8.0
pH: 7.85
Oxygen saturation (%), just below water surface: 88.2

Preliminary Experiment 2
Origin: Great Ouse River, Huntingdon, Cambridgeshire, UK
Date of sampling: 07 May 2019
Water body type: River
Appearance: Clear, yellow in colour
Sampling depth (cm): 117
Temperature (°C), just below water surface: 12.6
pH: 9.04
Oxygen saturation (%), just below water surface: 154.6

Preliminary Experiment 3
Origin: Great Ouse River, Huntingdon, Cambridgeshire, UK
Date of sampling: 09 August 2019
Water body type: River
Appearance: Clear, yellow in colour
Sampling depth (cm): 210
Temperature (°C), just below water surface: 20.7
pH: 7.94
Oxygen saturation (%), just below water surface:83.5
Details on source and properties of sediment:
N/A
Details on inoculum:
N/A
Duration of test (contact time):
< 7 d
Initial conc.:
ca. 100 µg/L
Based on:
test mat.
Details on study design:
Preliminary Experiment One
Portions of filtered surface water (100 mL) were added to cylindrical 500 mL glass bottles.
Single test vessels were established for three time-points: immediately after dose application, and after 2 and 7 days of incubation.
Air was drawn through each system at a flow rate of approximately 50 mL/minute.
All test systems were stirred continuously to facilitate oxygen transfer and maintained in darkness at approximately 12 ± 2°C in a temperature-controlled room.

Preliminary Experiments Two and Three
Portions of filtered surface water (100 mL) were added to cylindrical 250 mL (preliminary experiment three) or 500 mL (preliminary experiment two) glass bottles.
For preliminary experiment two, single test vessels were established for three time-points: immediately after dose application, and after 4 and 24 hours of incubation.
For preliminary experiment three, a single test vessel was established and sampled after 7 days of incubation.
All test systems were stirred continuously to facilitate oxygen transfer and maintained in darkness at approximately 12 ± 2°C in a temperature-controlled room.
For preliminary experiment two, the air flow rate was maintained at 5 mL/minute.
For preliminary experiment three, the test vessel was purged with air at a flow rate of 50 mL/minute during acclimatisation overnight then sealed (using a tap system either side of the headspace connecter) immediately after dose application. At sampling, the sealed test vessel was purged with air at a flow rate of 10 mL/minute for 60 minutes into a separate gas flow-through system arranged in series, as described above.

Sample Analysis
For all extractions and/or rinses, duplicated weighed aliquots (1.0 mL) were analysed by LSC. The volumes of all trapping solutions were measured and duplicate aliquots (1.0 mL) taken for LSC.
All tubing extracts for preliminary experiments one and three were stored at -20 ± 10°C prior to analysis. The tubing extracts for preliminary experiment two were analysed immediately after extraction.
Reference substance:
not specified
Compartment:
other: Surface Water Preliminary Experiment One
% Recovery:
82.3
Remarks on result:
other:
Remarks:
Time Zero
Compartment:
other: Surface Water Preliminary Experiment One
% Recovery:
2.6
Remarks on result:
other:
Remarks:
Day 7
Compartment:
other: Surface Water Preliminary Experiment Two
% Recovery:
90
Remarks on result:
other:
Remarks:
Time Zero
Compartment:
other: Surface Water Preliminary Experiment Two
% Recovery:
47.5
Remarks on result:
other:
Remarks:
24 hours
Compartment:
other: Surface Water Preliminary Experiment Three
% Recovery:
30.9
Remarks on result:
other:
Remarks:
Day 7
Remarks on result:
other: In the preliminary experiments, butylal or a rapidly formed degradation product volatised from the treated natural surface water. Therefore, no mineralization of butylal was observed.
Remarks on result:
other: In the preliminary experiments, butylal or a rapidly formed degradation product volatised from the treated natural surface water. Therefore, no mineralization of butylal was observed.
Transformation products:
not measured
Details on transformation products:
N/A
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
Preliminary Experiment One: The radioactivity recovered in the waters declined from 82.2% applied radioactivity (AR) at time zero to 2.2% AR by Day 2 and to 0.9% AR by Day 7. Low levels of volatile radioactivity were trapped in the 1 M potassium hydroxide solution at Day 2 (0.8% AR) and Day 7 (0.3% AR). Total radioactivity declined to 2.6% AR by Day 7. To investigate the low mass balances, all test vessels were rinsed with acetonitrile to recover any residual butylal. Little (<0.1% AR) or no radioactivity was recovered. Acetonitrile was added to the waters of the time zero and Day 2 samples to improve the solubility of butylal. However, the recovery of radioactivity decreased. Low levels (1.2% AR) of radioactivity were recovered from the tubing extracts at Day 7 suggesting butylal absorbed onto the plastic of the tubing. No radioactivity was recovered from the magnetic stirrer bar, glass adaptor head and fittings for the Day 7 sample. The O-ring insert for the vessel head for the Day 7 sample was extracted with acetonitrile. Low levels (0.2% AR) of radioactivity were recovered, suggesting that low levels of butylal absorbed to the plastic of the O-ring.
Chromatographic Analysis: The HPLC analysis of the time-zero showed no significant degradation of [14C]-butylal. Due to the low levels of radioactivity recovered, no HPLC analysis was conducted on the Day 2 and 7 waters from preliminary experiment one.

Preliminary Experiment Two: The amount of radioactivity recovered from the waters declined from 90.0% AR at time zero
to 0.6% AR after 24 hours. Volatile radioactivity increased up to 46.9% AR after 24 hours with 42.5% AR trapped in the chilled toluene. Radioactivity recovered from the plastic tubing up to 24 hours was 2.0 – 4.0% AR. Total radioactivity declined to 73.9% AR and 47.5% AR after 4 and 24 hours, respectively.
Chromatographic Analysis: No significant degradation of butylal was determined in either the time-zero or 4-hour waters where the proportion of [14C]-butylal was approximately 89.9% AR and 68.1% AR, respectively. Due to the low level of radioactivity recovered, no HPLC analysis was conducted on the 24-hour water from preliminary experiment two.

Preliminary Experiment Three: The amount of radioactivity recovered from the waters declined to 24.5% AR after 7 days. No volatile radioactivity was extracted from the charcoal cartridge and the polyurethane foam bung. No volatile radioactivity was trapped by ethylene glycol and the chilled toluene. Low (<0.3% AR) levels of radioactivity were trapped in the 1 M potassium hydroxide solution. The tubing between the test vessel and the humidifying test vessel was extracted with acetone/THF (1/1, v/v) and 6.1% AR was recovered.
Chromatographic Analysis: The proportion of [14C]-butylal in the Day 7 waters was approximately 18.5% AR with low levels (<5.6% AR%) of polar materials also detected.
Results with reference substance:
N/A

Recoveries of Radioactivity from Incubated Surface Water Treated with [14C]-Butylal (Preliminary Experiment 1)

            Days after application/Sample identity
 Time-zero/P01  Day 2/P02  Day 7/P03
 Waters  82.2 2.2  0.9 
 Investigations:      
 Test vessel rinse  0.1  0.1  nd
Adaptor head and magnetic stirrer rinse -  -  nd
 Tubing 1 extraction  -  1.2
 Tubing 2 extraction -  nd
 O-ring extraction 0.2 

Volatiles:

KOH

 - 0.8  0.3 
 Total recovery  82.3 3.1   2.6

Recoveries of Radioactivity from Incubated Surface Water Treated with [14C]-Butylal (Preliminary Experiment 2)

            Time after application/Sample identity
 Time-zero/P201  4 h/P202  24 h/P203
 Watersa  90.0  68.4  0.6
 Volatiles:      
  Ethylene glycol  -  1.3  0.7
  KOH  -  nd  0.2
  Toluene (chilled)  -  nd  42.5
  Carbon cartridgeb  -  nd  nd
 Polyurethane foam bungsb  -  0.2  1.5
 Tubing extractionsc  -  4.0  2.0
 Total volatiles  -  5.5  46.9
 Total recovery  90.0  73.9  47.5

Recoveries of Radioactivity from Incubated Surface Water Treated with [14C]-Butylal (Preliminary Experiment 3)

      Days after application/Sample identity
 Day 7/P301
 Watersa  24.5
Volatiles:   
  Ethylene glycol  nd
  KOH  0.3
  Toluene (chilled)  nd
 Carbon cartridged  nd
 Polyurethane foam bungsd  nd
 Tubing extractionsc  6.1
 Total volatiles  6.4
 Total recovery  30.9

All results are expressed as % applied radioactivity

nd not detected

a After the addition of 20% of acetonitrile (by volume)

b Extracted with acetonitrile (30 mL)

c Extracted with acetone/THF (1/1, v/v, 75 mL)

d Extracted with acetonitrile (20 mL)

Validity criteria fulfilled:
not applicable
Conclusions:
In the preliminary experiments, butylal or a rapidly formed degradation product volatised from the treated natural surface water. Therefore, no mineralization of butylal was observed.
Executive summary:

Three preliminary experiments were conducted to investigate the feasibility of conducting the main experiment of the mineralisation study. Natural surface water was treated with [14C]-butylal at a nominal application rate of 100 μg/L.

Preliminary experiment one: treated surface water samples were attached to air-flow lines with a flow rate of 50 mL/minute with traps to collect carbon dioxide and were incubated with continuous stirring at 12±2 °C in darkness for periods of up to 7 days.

Total recoveries of for the samples declined from 82.3% applied radioactivity (% AR) at time zero to 2.6% AR after 7 days. Direct volatile radioactivity as carbon dioxide (14CO2) accounted for a maximum of 0.8% AR at Day 2. Low (<1.4% AR) levels of radioactivity adhered to the plastic components of the test system.

 

Preliminary experiment two: treated surface water samples were attached to air-flow lines with a flow rate of 5 mL/minute with a selection of traps to collect organic volatiles and carbon dioxide and were incubated with continuous stirring at 12±2 °C in darkness for periods of up to 1 day.

Total recoveries of radioactivity for samples declined from 90.0% AR at time zero to 47.5% AR at 24 hours. Organic volatiles accounted for a maximum 46.9% AR whereas carbon dioxide (14CO2) accounted for a maximum of 0.2% AR.

Significant amounts (42.5% AR) of radioactivity were trapped in the chilled toluene with low levels (<2% AR) of radioactivity trapped by the ethylene glycol and polyurethane foam bungs or adhering to the plastic components of the test system.

Preliminary experiment three: one treated surface water sample was incubated with continuous stirring at 12±2 °C in darkness in a closed system for 7 days. After 7 days, the sample was flushed with air at a flow rate of 10 mL/minute for 1 hour prior to sampling.

The total recovery of radioactivity for the sample was 30.9% AR. Total volatiles accounted for 6.4% AR with low levels (<0.3% AR) recovered in the trapping media.

In conclusion, the results obtained from the preliminary experiments suggest that butylal or a rapidly formed degradation product volatised from the natural waters after short incubation periods (up to 7 days) at 12 ± 2°C. In addition, the applied radioactivity in the test vessel was not effectively recovered using the selected trapping media, which resulted in low mass balances for the preliminary experiments.

Based on the preliminary results, it was considered that the main experiment was not feasible.

Description of key information

The results obtained from the preliminary experiments suggest that butylal or a rapidly formed degradation product volatised from the natural waters after short incubation periods. Based on the preliminary results, it is considered that the main experiment was not feasible.

It is not feasible to conduct the full OECD 309 study as the test item did not remain in the natural water test system for sufficient time to allow mineralisation to carbon dioxide to take place. Therefore the identification of the degradation products of the test item in the conditions of the OECD 309 is not feasible.

Key value for chemical safety assessment

Additional information

Three preliminary experiments were conducted (Covance CRS Limited, 2019, Study CP42NS) to investigate the feasibility of conducting the main experiment of the mineralisation study. The study was performed according to OECD test guideline 309 and in compliance with GLP.

 

Natural surface water was treated with [14C]-butylal at a nominal application rate of 100 μg/L. All water sample was incubated with continuous stirring at 12 ± 2 °C in darkness in a closed system.

 

In the preliminary experiment one the treated surface water samples were attached to air-flow lines with a flow rate of 50 mL/minute with traps to collect carbon dioxide and were incubated for periods of up to 7 days. Total recoveries of for the samples declined from 82.3% applied radioactivity (% AR) at time zero to 2.6% AR after 7 days. Direct volatile radioactivity as carbon dioxide (14CO2) accounted for a maximum of 0.8% AR at Day 2. 1.4% AR levels of radioactivity adhered to the plastic components of the test system.

 

In the preliminary experiment two the treated surface water samples were attached to air-flow lines with a flow rate of 5 mL/minute with a selection of traps to collect organic volatiles and carbon dioxide and were incubated for periods of up to 1 day. Total recoveries of radioactivity for samples declined from 90.0% AR at time zero to 47.5% AR at 24 hours. Organic volatiles accounted for a maximum 46.9% AR whereas carbon dioxide (14CO2) accounted for a maximum of 0.2% AR. 42.5% AR of radioactivity were trapped in the chilled toluene with 2% AR of radioactivity trapped by the ethylene glycol and polyurethane foam bungs or adhering to the plastic components of the test system.

In the preliminary experiment three one treated surface water sample was incubated in a closed system for 7 days. After 7 days, the sample was flushed with air at a flow rate of 10 mL/minute for 1 hour prior to sampling. The total recovery of radioactivity for the sample was 30.9% AR. Total volatiles accounted for 6.4% AR with <0.3% AR recovered in the trapping media.

 

The results obtained from the preliminary experiments suggest that butylal or a rapidly formed degradation product volatised from the natural waters after short incubation periods (up to 7 days). In addition, the applied radioactivity in the test vessel was not effectively recovered using the selected trapping media, which resulted in low mass balances for the preliminary experiments.

Based on the preliminary results, it is considered that the main experiment was not feasible.