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Environmental fate & pathways

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:
2013-02-15 to 2013-06-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 309 but was not performed according to GLP as this study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes. This study is well documented and is considered reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Version / remarks:
2004
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
This study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes.
Specific details on test material used for the study:
No additional information
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Sediments and associated water from the Brandywine Creek (Chadds Ford, Pennnsylvania, USA) were used in the study. Sediment samples were taken from the entire 2 cm upper layer of the underwater sediment.
- Storage conditions: Freshly collected surface water and sediment were stored refrigerated.
Surface water:
- pH at time of collection: 6.0
- Temperature (°C) at time of collection: 3.5 °C
- Dissolved oxygen concentration: 8.2 mg/L
- Organic carbon concentration: 1.8 mg C/L
- Organic carbon (%): 4.74 % (sediment)
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Sediment and associated water were combined at a ratio of approximately 40 g (wet weight): 250 mL and stirred vigorously. The mixture was allowed to stand for approximately 5 minutes after which the sediment amended water layer above the settled solids was decanted. The total suspended solids concentration of the sediment amended water layer was measured and adjusted to approximately 1 g/L. The standard plate count proved microbial presence (average = 1.81×10^4 cfu/mL).
- Sediment samples sieved: Yes; the sediment was separated from the water by filtration and wet-sieved using a 2 mm-sieve.
Details on inoculum:
Not applicable
Duration of test (contact time):
56 d
Initial conc.:
10 µg/L
Based on:
test mat.
Initial conc.:
50 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: Test volume was 2 L for the biotic treatments and 1 L for the sterile and reference controls
- Test temperature: approximately 20 °C (±2 °C)
- pH adjusted: Yes; adjusted with 1.5 N KOH to match that of the live system (7.2)
- Suspended solids concentration: 17 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Test was conducted in flow-through mode where the chambers were actively aerated at a rate of approximately 5 mL/minute. The biotic test chambers were connected to a gas trapping system consisting of a polyurethane plug (PUP) followed by a series CO2 traps containing an alkali solution. The sterile chambers were connected to a gas trapping system consisting of a polyurethane plug (PUP). The reference control chambers were connected to a gas trapping system consisting of series CO2 traps containing an alkali solution.
- Number of culture flasks/concentration: Triplicate biotic and single abiotic (sterile) test chambers were prepared for each concentration of test substance. In addition, duplicate reference control test chambers were prepared.
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used: The conversion of radiolabeled test substance to 14CO2 (carbon dioxide) was measured by trapping 14CO2 in base traps and quantifying dissolved 14CO2. Volatilized test material was collected on a polyurethane foam plug and quantified.

SAMPLING
- Sampling frequency: Samples for LSC counting and analytical determination of test substance concentration were removed from the biotic and abiotic treatments at approximately 5 minutes then on days 2, 5, 7, 14, 21, 28 and 56. The 14CO2 traps were removed for analysis from the biotic and reference control gas trapping systems on days 2, 5, 7, 14, 21, 28, 42 and 56. In addition, samples for determination of dissolved CO2 were removed from the biotic and reference control chambers on days 2, 5, 7, 14, 21, 28, 42 and 56. The polyurethane plugs (volatile trap) were removed for analysis on days 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 and 56. Prior to sampling, the test vessels were placed on a magnetic stirrer and the contents were mixed thoroughly. Samples were removed using 50 mL plastic syringes.
- Determining Loss Due To Volatilization: At each specified sampling interval, the polyurethane plugs (PUP) were exchanged with a new plug. The used (PUP) was then extracted with a known volume of the solvent system used to extract the parent and metabolite(s).
- Radioactivity Remaining with the Solids: The extracted solids on the glass fiber filters were combusted to determine the total
radioactivity remaining with the solids.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Yes
- Biotic positive control: Yes

STATISTICAL METHODS:
- Regression analyses of the percentage of the test substance and total metabolites as a function of time were performed using Sigma Plot software 2011.
Reference substance:
aniline
Remarks:
radiolabeled 58 µg/L
Test performance:
No data
Key result
% Degr.:
> 32 - < 35
Parameter:
radiochem. meas.
Sampling time:
56 d
Remarks on result:
other: at 10 and 50 μg/L, respectively.
Key result
Compartment:
sediment
DT50:
0.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: At 10 µg/L
Key result
Compartment:
sediment
DT50:
0.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: At 50 µg/L
Transformation products:
no
Details on transformation products:
Not applicable
Evaporation of parent compound:
no
Volatile metabolites:
yes
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MINERALISATION
- The extent of mineralization or ultimate degradation ranged from 32-35 % for test systems dosed at 10 and 50 μg/L, respectively.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Volatilized test material collected on a polyurethane foam plugs from the biotic replicates at both tested concentrations represented <10% of the initial activity. Volatilized test material collected from abiotic treatments was minimal and reached a maximum of 1.2% for both tested concentrations at the termination of the test on day 56.

- Rad-TLC analysis: Rad-TLC analysis of the test item standard revealed two distinct regions (Rf 0.02 and 0.46). The region (Rf 0.46) was associated with the parent test material and was present at 48.1 % of the initial activity at the beginning of the study. Rad-TLC analysis of the solvent extracts from the biotic replicates and abiotic treatments resulted in a similar disappearance pattern of the parent test substance, at both tested concentrations.
- The average 14CO2 production for the biotic replicates dosed at 10 and 50 µg/L was 35.0 and 32.1% on day 56, respectively.
- For the biotic replicates, the level of activity associated with the solids increased over time but never exceeded 15 % of dose for both tested concentrations. For the abiotic treatments dosed at 10 and 50 µg/L, the level of activity associated with the solids was significant and reached a maximum of 77.0 and 65.5%, respectively.
- Regression analyses of the percentage of the test substance and total metabolites as a function of time were performed. The loss of parent DT50 and DT90 were calculated using a single three-parameter decay curve and were approximately 0.9 and 2.9 days at 10 µg/L and 0.8 and 2.6 days at 50 µg/L. The elimination rate for the parent compound is a result of primary and ultimate degradation as well as potential chemical hydrolysis, as the abiotic controls suggest.
Results with reference substance:
The average 14CO2 production from the positive control replicates dosed with 14C-Aniline, was 17.9% on day 56. The standard plate count proved microbial presence; however the results indicate partial degradation of the reference material. A half-life for aniline of 2.3 days has been reported, yet in an industrial river. Therefore, degradation of aniline could have been impacted by the test matrix since the sediment water utilized in the test was collected from the area where there is no potential pollution source.

None

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the extent of mineralization or ultimate degradation ranged from 32 to 35 % for test systems dosed at 10 and 50 μg/L, respectively.
Executive summary:

The biodegradability of the test item has been determined by the Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test according to the OECD Guideline No. 309.

Test systems were dosed with14C-labeled test substance and incubated at approximately 20 ± 2 ºC for 56 days. The test substance was dosed to both biotic and abiotic test vessels at a nominal concentration of 10 and 50 µg/L. The disappearance of parent test substance and the formation of metabolites were followed using radio thin layer chromatography (Rad-TLC). The conversion of radiolabeled test substance to 14CO2 (carbon dioxide) was measured by trapping 14CO2 in base traps and quantifying dissolved 14CO2. Volatilized test material was collected on a polyurethane foam plug and quantified when appropriate. The test system was water amended with sediment at approximately 1g/L. The abiotic control was identical to the biologically active treatment with the exception that it was amended at a nominal concentration of 1 g/L with mercuric chloride. A reference substance (with14C-Aniline at a nominal concentration of 58 µg/L) was used to assess the microbial activity of the test system. Samples were removed from the test vessels over a 56-day period.

The data shows rapid disappearance of the parent test material from the sediment water system at both tested concentrations. The DT50 and DT90 values for test item were calculated using a single three-parameter decay curve and were approximately 0.9 and 2.9 days at 10 µg/L and 0.8 and 2.6 days at 50 µg/L, respectively. The elimination rate for the parent compound is a result of primary and ultimate degradation as well as potential chemical hydrolysis. The extent of mineralization or ultimate degradation ranged from 32 to 35 % for test systems dosed at 10 and 50 μg/L, respectively. The viability controls in which the sediment water was incubated with radiolabeled aniline, demonstrated the presence of microbial activity, however went partial ultimate degradation.

Description of key information

OECD Guideline 309, non-GLP, Key study, validity 2:

Rapid disappearance of the parent test substance from the sediment water system at both tested concentrations (primary and ultimate degradation as well as potential chemical hydrolysis):

DT50 (20°C, 10 µg/L) = 0.9 days;

DT50 (20°C, 50 µg/L) = 0.8 days.

The extent of mineralization or ultimate degradation ranged from 32 to 35 % for test systems dosed at 10 and 50 μg/L, respectively, after 56 days.

Key value for chemical safety assessment

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

Additional information

To assess the biodegradability of the registered substance and the formation of metabolites in sediment water system, one valid study is available.

This study (Wildlife International, 2013), assessed as the key study, was performed on the registered substance according to OECD Guideline 309 but without GLP compliance as this study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes. Test systems were dosed with14C-labeled test substance and incubated at approximately 20 ± 2 ºC for 56 days. The test substance was dosed to both biotic and abiotic test vessels at a nominal concentration of 10 and 50 µg/L. The disappearance of parent test substance and the formation of metabolites were followed using radio thin layer chromatography (Rad-TLC). The conversion of radiolabeled test substance to 14CO2(carbon dioxide) was measured by trapping 14CO2in base traps and quantifying dissolved 14CO2. Volatilized test material was collected on a polyurethane foam plug and quantified when appropriate. The test system was water ammended with sediment at approximately 1g/L. The abiotic control was identical to the biologically active treatment with the exception that it was amended at a nominal concentration of 1 g/L with mercuric chloride.A reference substance (with14C-Aniline at a nominal concentration of 58 µg/L) was used to assess the microbial activity of the test system. Samples were removed from the test vessels over a 56-day period.

The data shows rapid disappearance of the parent test substance from the sediment water system at both tested concentrations. The DT50 and DT90 values for test substance were calculated using a single three-parameter decay curve and were approximately 0.9 and 2.9 days at 10 µg/L and 0.8 and 2.6 days at 50 µg/L, respectively. The elimination rate for the parent compound is a result of primary and ultimate degradation as well as potential chemical hydrolysis. The extent of mineralization or ultimate degradation ranged from 32 to 35 % for test systems dosed at 10 and 50 μg/L, respectively. The viability controls in which the sediment water was incubated with radiolabeled aniline, demonstrated the presence of microbial activity, however underwent only partial ultimate degradation.