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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2019 - XXXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Deviations:
no
GLP compliance:
yes
Remarks:
Exception: No claim of GLP Compliance is made for the aqueous sediment collection data or the kinetic evaluation.
Specific details on test material used for the study:
Radiolabeled test material: 4,4’-methylene bis(dibutyldithiocarbamate), [CH2-14C]
Non-Radiolabeled test material (for co-chromatography): 4,4’-methylene bis(dibutyldithiocarbamate)
Radiolabelling:
yes
Remarks:
The test material, [14C]-4,4’-methylene bis(dibutyldithiocarbamate), was applied to the test system without radiodilution.
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
- Details on collection: Aerobic and anaerobic aquatic sediments from Calwich Abbey Lake (Calwich, Staffordshire, UK) and Emperor Lake (Chatsworth, Derbyshire, UK) were provided by LRA Labsoil (Derby, UK)
- Sampling depth: 40-45 cm (Calwich); 30-50 cm (Emperor)
- Storage conditions: Shipped in separate container from sediment (with the exclusion of oxygen for the anaerobic systems) and, upon receipt at the testing facility, stored at +5 ± 3°C.
- Temperature (°C) at time of collection: 12.7 (Calwich); 15.3 (Emperor)
- pH at time of collection: Aerobic: 8.40 (Calwich); 6.83 (Emperor)
- Oxygen concentration (in water): 115.0% ASV (Calwich); 103.5% ASV (Emperor)
- Hardness (CaCO3): Aerobic: 260 (Calwich); 46.4 (Emperor); Anaerobic: 258 (Calwich); 42.0 (Emperor)
- Total organic carbon: Aerobic: 5.43 mg/L (Calwich); 8.46 mg/L (Emperor); Anaerobic: 5.76 mg/L (Calwich); 9.09 mg/L (Emperor)
- Filtration: 0.2 mm sieve
Details on source and properties of sediment:
- Details on collection: Aerobic and anaerobic aquatic sediments from Calwich Abbey Lake (Calwich, Staffordshire, UK) and Emperor Lake (Chatsworth, Derbyshire, UK) were provided by LRA Labsoil (Derby, UK)
- Sampling depth: 5 (aerobic); >5 cm anaerobic
- Storage conditions: Shipped in separate container from water (with the exclusion of oxygen for the anaerobic systems) and, upon receipt at the testing facility, stored at +5 ± 3°C.
- Textural classification: Aerobic: Silty clay loam (Calwich); Sandy clay loam (Emperor); Anaerobic: Silt loam (Calwich); Sandy clay loam (Emperor)
- pH at time of collection: Aerobic: 8.40 (Calwich); 6.83 (Emperor); Anaerobic: 7.8 (Calwich); 5.9 (Emperor)
- Organic carbon: Aerobic: 4.89% (Calwich); 1.91% (Emperor); Anaerobic: 4.80% (Calwich); 1.99% (Emperor)
- Redox potential: Aerobic: < -100 mV; Anaerobic: < -150 mV
- Oxygen concentration (at water/sediment interface): Aerobic: 114.7% ASV (Calwich); 101.7% ASV (Emperor)
- CEC (meq/100 g): Aerobic: 15.4 (Calwich); 15.4 (Emperor); Anaerobic: 19.6 (Calwich); 15.8 (Emperor)
- Sediment samples sieved: 2 mm sieve
Details on inoculum:
Microbiological analyses of sediment and water were performed at the start and end of the incubation period. Both aquatic sediments were microbiologically active throughout the incubation period and there were no appreciable differences in levels of the various organisms at the end of the incubation period between untreated samples and samples treated with solvent.
Duration of test (contact time):
100 d
Initial conc.:
0.1 mg/L
Based on:
other: Application rate calculated from radioactivity and volume applied, and specific activity of the test material.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
Details on study design:
Test material was applied to the water layer of samples of two aquatic sediment types (Calwich Abbey Lake and Emperor Lake) at a nominal rate of 0.1 mg/L. The volume ratio of wet sediment to water in each sample was between 1:3 and 1:4. Test vessels were to cylindrical one-litre glass bottles of approximately 9.5 cm internal diameter. Aquatic sediment systems were acclimatized separately under aerobic and anaerobic conditions prior to test item application until reasonable stability was established with respect to the pH, oxygen concentration and redox potential in the water and the pH and redox potential in the sediment.

Vessels were arranged in flow-through systems designed to trap volatile radiolabeled compounds including 14CO2. The systems were incubated in darkness at 12 +/- 2 deg C in a temperature-controlled room for up to 100 days. For the aerobic phase, air was drawn through each system at a flow rate of approximately 50 mL/minute. For the anaerobic phase, nitrogen was pushed through each system at flow rates of approximately 50-70 mL/minute. Flow rates were checked and adjusted throughout the incubation period.

Vessels assigned for system parameter measurements were used for the measurement of pH and redox potential in both phases and oxygen content in the water phase. Measurements were made twice weekly (aerobic vessels) or weekly (anaerobic vessels) throughout the acclimatization period and then, following test material application, at every sampling interval.

Additional vessels were established for the determination of the microbiological activity at the start and end of the incubation period and the measurement of the pH and redox potential in both phases and oxygen content in the water phase. Additional vessels were established for the determination of the microbiological activity at the start and end of the incubation period and the measurement of the pH and redox potential in both phases and oxygen content in the water phase.

Vessels were also established to determine the extent of methanogenesis from the aquatic sediments, although methanogenesis analysis was not required since mass balances remained above 90% of applied radioactivity.

Duplicate samples of each aquatic sediment and incubation condition were taken for analysis immediately after application and after 2, 7, 14, 30, 58 or 57 (aerobic or anaerobic) and 100 days of incubation.
Trapping solutions were taken for analysis with the associated samples at sampling. Trapping solutions associated with all remaining vessels were taken for analysis and replaced at 14, 30, 58 or 57 (aerobic or anaerobic) and 100 days after application.

A kinetic analysis was performed to evaluate the decline of parent test material in the water and sediment phase of each aquatic sediment, and in the total systems.
Compartment:
natural water / sediment: freshwater
Remarks on result:
other: 92.7 to 101.5% applied radioactivity
Compartment:
natural water: freshwater
DT50:
5.03 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Remarks:
aerobic
Compartment:
natural sediment: freshwater
DT50:
406 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Remarks:
aerobic
Key result
Compartment:
natural water / sediment: freshwater
DT50:
240 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Remarks:
aerobic
Compartment:
natural water: freshwater
DT50:
46.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Remarks:
anaerobic
Compartment:
natural sediment: freshwater
Temp.:
12 °C
Remarks on result:
not determinable
Remarks:
Calwich Abbey Lake / anaerobic
Key result
Compartment:
natural water / sediment: freshwater
DT50:
958 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Remarks:
anaerobic
Compartment:
natural water: freshwater
DT50:
6.67 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
aerobic
Compartment:
natural sediment: freshwater
DT50:
226 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
aerobic
Key result
Compartment:
natural water / sediment: freshwater
DT50:
215 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
aerobic
Compartment:
natural water: freshwater
DT50:
39 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
anaerobic
Compartment:
natural sediment: freshwater
DT50:
1 390 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
anaerobic
Key result
Compartment:
natural water / sediment: freshwater
DT50:
1 660 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Remarks:
anaerobic
Transformation products:
not specified
Remarks:
; a pathway for the transformation of the parent material in aerobic surface water will be proposed based upon completion of the the degradation identification by LC-MS (in progress).
Details on transformation products:
HPLC analysis of the overlying water and extracts of sediment resolved up to 16 components in addition to the parent test material, only one of which constituted a major degradate on the basis that it accounted for more than 5% applied radioactivity at two or more sequential sampling intervals.

In the aerobic aquatic sediment systems, the test material was degraded to Unknown 1 (up to a mean of 6.8% applied radioactivity in the total system after 100 days). In addition, the test material was degraded to up to 12 low level unidentified degradates, none of which exceeded 2.9% in the total system on any one sampling interval. In the anaerobic aquatic sediment systems, the test material was degraded to up to eight low level unidentified, none of which exceeded 2.8% in the total system on any one sampling interval.
Evaporation of parent compound:
no
Volatile metabolites:
yes
Residues:
yes
Details on results:
CHARACTERIZATION OF THE TEST SYSTEM
Both aquatic sediment systems were microbiologically active throughout the incubation period and there were no appreciable differences in levels of the various organisms at the end of the incubation period between untreated samples and samples treated with solvent. During the incubation period for the aerobic samples, oxygen levels in the water (in the range 68.2 to 112.2% air saturated value) and redox potentials in the water (generally greater than +400 mV) and sediment (generally less than -100 mV) were indicative of an aerobic, oxidising water phase and a reducing sediment phase. During the incubation period for the anaerobic samples, oxygen levels in the water (less than 3% saturation) and redox potentials in the water (generally less than +270 mV) and sediment (generally less than -150 mV) were indicative of an anaerobic, reducing water phase and sediment phase. The temperature of the room remained within the range 12 +/- 2 deg C throughout the incubation period.

RECOVERY AND DISTRIBUTION OF RADIOACTIVITY
The mean total recoveries of radioactivity (mass balance) for both aquatic sediments incubated under aerobic or anaerobic conditions were in the range 92.7 to 101.5% applied radioactivity (AR).

Calwich Abbey Lake aquatic sediment (aerobic)
The mean radioactivity in the water layer declined from 95.7% AR at time zero to 3.7% AR after 100 days of incubation. In sediment, the total radioactivity increased to a mean of 91.7% AR after 30 days and remained at a similar level after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a maximum mean of 7.3% AR after 7 days and then remained at a similar level up to 100 days. Volatile radioactivity accounted for a maximum mean of 3.5% AR after 100 days.

Emperor Lake aquatic sediment (aerobic)
The mean radioactivity in the water layer declined from 92.0% AR at time zero to 4.0% AR after 100 days of incubation. In sediment, the total radioactivity increased to a mean of 89.3% AR after 58 days and remained at a similar level after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a maximum mean of 8.5% AR after 100 days. Volatile radioactivity accounted for a maximum mean of 4.1% AR after 100 days.

Calwich Abbey Lake aquatic sediment (anaerobic)
The mean radioactivity in the water layer declined from 90.3% AR at time zero to 24.8% AR after 100 days of incubation. In sediment, the total radioactivity increased to a mean of 70.6% AR after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a maximum mean of 5.6% AR after 7 days and then remained at a similar level up to 100 days. Volatile radioactivity accounted for a maximum mean of 1.2% AR after 100 days.

Emperor Lake aquatic sediment (anaerobic)
The mean radioactivity in the water layer declined from 86.0% AR at time zero to 21.6% AR after 57 days and remained at a similar level after 100 days of incubation. In sediment, the total radioactivity increased to a mean of 73.8% AR after 57 days and remained at a similar level after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a maximum mean of 7.4% AR after 30 days and remained at a similar level after 100 days. Volatile radioactivity accounted for a maximum mean of 1.9% AR after 100 days.

CHROMATOGRAPHIC ANALYSIS
Proportions of parent test material and its radioactive degradates were determined by HPLC analysis for the overlying water, sediment extracts and total systems for the aerobic and anaerobic aquatic systems.

In the total aquatic sediment system of Calwich Abbey Lake incubated under aerobic conditions, parent test material declined from a mean of 94.4% AR at time zero to 71.8% AR after 100 days of incubation. In the total aquatic sediment system of Emperor Lake incubated under aerobic conditions, parent test material declined from a mean of 96.4% AR at time zero to 66.4% AR after 100 days of incubation.

In the total aquatic sediment system of Calwich Abbey Lake incubated under anaerobic conditions, parent test material declined from a mean of 96.0% applied radioactivity at time zero to 86.1% AR after 100 days of incubation. In the total aquatic sediment system of Emperor Lake incubated under anaerobic conditions, 4 parent test material declined from a mean of 96.3% AR at time zero to 86.7% applied radioactivity after 100 days of incubation.

IDENTIFICATION OF RADIOACTIVE COMPONENTS
The identity of radiolabeled test material applied was established by HPLC co-chromatographic correspondence with non-radiolabeled reference material by co-injection.

TRANSFORMATION PATHWAY
A pathway for the transformation of the parent material in surface water will be proposed based upon the degradation identification by liquid chromatography with mass spectrometry (LC-MS).
Validity criteria fulfilled:
yes
Conclusions:
The parent material dissipated rapidly from the water of aquatic sediment systems incubated under aerobic conditions, with estimated DT50 values of 5.0 days (Calwich Abbey Lake) and 6.7 days (Emperor Lake). Decline in the overall system incubated under aerobic conditions corresponded to estimated DT50 values of 240 days (Calwich Abbey Lake) and 215 days (Emperor Lake).

The parent material dissipated slowly from the water of aquatic sediment systems incubated under anaerobic conditions, with estimated DT50 values of 46 days (Calwich Abbey Lake) and 39 days (Emperor Lake). Decline in the overall system incubated under anaerobic conditions corresponded to estimated DT50 values of 958 days (Calwich Abbey Lake) and 1660 days (Emperor Lake).

The parent material was degraded to one major degradate, Unknown 1 (up to a mean of 6.8% after 100 days) in the total system and 15 low level unidentified degradates (mean recovery did not exceed 2.9% applied radioactivity for any one minor degradate on any sampling occasion), was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide.
Executive summary:

The fate of the test material was studied in two natural aquatic sediment systems under laboratory conditions according to OECD Guideline 308. The sediment from Emperor Lake was a sandy clay loam with an acidic pH and low organic carbon content, while that from Calwich Abbey Lake was a neutral silt loam with a higher organic carbon content. Samples of each aquatic sediment system were allowed to acclimatize separately under aerobic or anaerobic conditions before being treated with radiolabelled test material at a rate of 0.1 mg/L based on the amount of water in the test vessel including that present within the sediment. The samples were incubated under aerobic or anaerobic conditions at about 12 deg C in darkness for periods of up to 100 days.

 

Mean total recoveries of radioactivity (mass balances) for both aquatic sediments incubated under aerobic or anaerobic conditions were between 92.7% and 101.5% applied radioactivity. 

 

In Calwich Abbey Lake aquatic sediment incubated under aerobic conditions, the radioactivity in the water layer declined from a mean of 95.7% applied radioactivity at time zero to 3.7% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 91.7% applied radioactivity after 30 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sedimentaccounted for a mean of ≤7.3% applied radioactivitythroughout. Volatile radioactivity accounted for a maximum mean of 3.5% applied radioactivity after 100 days.

 

In Emperor Lake aquatic sediment incubated under aerobic conditions, the radioactivity in the water layer declined from a mean of 92.0% applied radioactivity at time zero to 4.0% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 89.3% applied radioactivity after 58 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤8.5% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 4.1% applied radioactivity after 100 days.

 

In Calwich Abbey Lake aquatic sediment incubated under anaerobic conditions, the radioactivity in the water layer declined from a mean of 90.3% applied radioactivity at time zero to 24.8% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 70.6% applied radioactivity after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤5.6% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 1.2% applied radioactivity after 100 days.

 

In Emperor Lake aquatic sediment incubated under anaerobic conditions, the radioactivity in the water layer declined from a mean of 86.0% applied radioactivity at time zero to 21.6% after 57 days and remained at a similar level up to 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 73.8% applied radioactivity after 57 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤7.4% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 1.9% applied radioactivity after 100 days.

 

DT50 and DT90 values were estimated using single first order kinetics modelling based on the observed degradation of the parent test material from the water, the sediment and from the total aquatic sediment system at approximately 12 deg C. In the two aerobic systems, DT50 values were determined to be 5.03 - 6.67 days (water), 226 - 406 days (sediment), and 215 - 240 days (whole system), and DT90 values ranged from 16.7 - 22.2 days (water), 749 - 1350 days (sediment), and 713 - 799 days (whole system). In the two anaerobic systems, DT50 values were determined to be 39 - 46.4 days (water), 1390 days (sediment), and 958 - 1660 days (whole system), and DT90 values ranged from 130 - 154 days (water), 4610 days (sediment), and 3180 - 5500 days (whole system). The DT values for sediment were estimated only for Emperor Lake only, as there was no decline in parent material in Calwich Abbey Lake sediments.

The test material was degraded to one major degradate, Unknown 1 (up to a mean of 6.8% in the total system) and 15 low level unidentified degradates (mean recovery did not exceed 2.9% applied radioactivity for any one minor degradate on any sampling occasion), was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 October 2019 - XXXX
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:
no
GLP compliance:
yes
Specific details on test material used for the study:
Radiolabeled test material: 4,4’-methylene bis(dibutyldithiocarbamate), [CH2-14C]
Non-Radiolabeled test material (for co-chromatography): 4,4’-methylene bis(dibutyldithiocarbamate)
Radiolabelling:
yes
Remarks:
The test material, [14C]-4,4’-methylene bis(dibutyldithiocarbamate), was applied to the test system without radiodilution.
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on source and properties of surface water:
- Source: River Waveney, Diss, Norfolk, UK
- Sampling depth (cm): 5
- Storage conditions: Surface water was collected and transported in containers to the testing facility and prior to use on the day of collection, was passed through a 0.1 mm sieve.
- Temperature (°C) at time of collection: 7.1
- pH at time of collection: 7.41
- Oxygen saturation (%), just below water surface: 81.9
- Appearance: Clear, with yellow tinge
- Hardness (CaCO3): 428
- Total organic carbon (mg/L): 12.6
- Dissolved organic carbon (mg/L) 12.9
- Water filtered: yes (100-μm nylon filter or a coarse paper filter)
Details on inoculum:
The sterile control vessels were incubated under study conditions for approximately 5 hours prior to test item application. All other test vessels were incubated under study conditions overnight prior to test item application to enable the microbial organisms to acclimatize to study conditions.

At the beginning and end of the incubation period, samples of surface water were analyzed for aerobic bacteria, aerobic bacterial spores, actinomycetes and fungi.
Duration of test (contact time):
62 d
Initial conc.:
10.5 µg/L
Based on:
test mat.
Remarks:
Based on total radioactivity of the application solution.
Initial conc.:
101 µg/L
Based on:
test mat.
Remarks:
Based on total radioactivity of the application solution.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
Details on study design:
Preliminary experiments were performed with surface water to establish the analytical methodology and confirm the sampling schedule for the main experiment.

In the main experiment, surface water was treated with [14C]-4,4’-methylene bis(dibutyldithiocarbamate) at nominal application rates of 10 µg/L and 100 µg/L. Treated surface water samples were attached to air flow lines with traps to collect carbon dioxide and incubated with continuous stirring to maintain aerobic conditions at 12 +/- 2 deg C in darkness for periods of up to 62 days.

Prior to use, the surface water was filtered through a 100-μm nylon filter or a coarse paper filter. Portions of filtered surface water (100 mL) were added to cylindrical 500 mL glass bottles. Duplicate flasks were prepared for seven sampling intervals (plus two spare flasks). Duplicate controls (sampled at 62 days) were sterilized by autoclaving prior to addition of the test material. Duplicate reference control vessels containing [14C]-benzoic acid (10 μg/L, with and without solvent) were included to confirm minimum microbial activity and to determine possible adverse effects of solvent. Five vessels of surface water were set up for microbiological analysis and two vessels were set up for measurements of pH and oxygen content. These vessels were not treated with test material.

Vessels established for treatment with the test or reference material were incorporated into individual flow-through systems in series after a humidifying vessel containing water to humidify the air-flow and before two vessels containing 1 M aqueous potassium hydroxide solution (KOH) with phenolphthalein indicator to trap and measure 14CO2, concluding with a non-return valve to prevent accidental backflow through the test apparatus.

Air was drawn through each system at a flow rate of approximately 50 mL/minute. Flow rates were checked at the beginning of acclimatization, at test item application and at intervals thereafter, and adjusted throughout the incubation period.

The samples assigned for microbiological activity determination and system parameter measurements were connected, in series, into separate flow-through systems. Humidified air was passed through these vessels, but no traps were included.

Throughout the incubation period, the samples assigned for system parameter measurements were used for the measurement of pH and oxygen content in the surface water at every sampling interval.

All test systems were stirred continuously to facilitate oxygen transfer and maintained in darkness at approximately 12 +/- 2 deg C in a temperature-controlled room.

For each test concentration, duplicate samples of surface water were taken for analysis immediately after test item application and after 2, 7, 13, 29, 43 and 62 days of incubation. Duplicate sterile controls were taken for analysis after 62 days of incubation. Trapping media were taken for analysis with the associated samples at sampling. In addition, trapping media associated with all remaining vessels were taken for analysis and replaced with fresh trapping media after 13 days and subsequently at approximately two-weekly intervals, to coincide with vessel sampling intervals.
Reference substance:
benzoic acid, sodium salt
Compartment:
natural water: freshwater
% Recovery:
83.2
Remarks on result:
other: Day 62 (total range: 82.2 - 99.4%)
Remarks:
(applied radioactivity; 10 ug/L treatment)
Compartment:
natural water: freshwater
% Recovery:
87.2
Remarks on result:
other: Day 62 (total range: 80.4 - 100.4%)
Remarks:
(applied radioactivity; 100 ug/L treatment)
Compartment:
abiotic control measured at end of test
% Recovery:
89.2
Remarks on result:
other:
Remarks:
(Day 62, mean % applied radioactivity; 10 ug/L treatment)
Compartment:
abiotic control measured at end of test
% Recovery:
96.8
Remarks on result:
other:
Remarks:
(Day 62, mean % applied radioactivity; 100 ug/L treatment)
Key result
% Degr.:
88.5
Parameter:
radiochem. meas.
Remarks:
(10 ug applied, nominal)
Sampling time:
62 d
Remarks on result:
other:
Remarks:
(as net change in parent, % applied radioactivity)
Key result
% Degr.:
91
Parameter:
radiochem. meas.
Remarks:
(100 ug applied, nominal)
Sampling time:
43 d
Remarks on result:
other:
Remarks:
(as net change in parent, % applied radioactivity)
Key result
Compartment:
natural water: freshwater
DT50:
10.3 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other:
Remarks:
(10 ug/L; nominal applied concentration)
Key result
Compartment:
natural water: freshwater
DT50:
16 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other:
Remarks:
(100 ug/L; nominal applied concentration)
Other kinetic parameters:
first order rate constant
Transformation products:
not specified
Remarks:
; a pathway for the transformation of the parent material in aerobic surface water will be proposed based upon completion of the the degradation identification by LC-MS (in progress).
Details on transformation products:
The major degradates designated as Unknown 1 (RT 28.9 min), Unknown 3 (RT 27.0 min) and Unknown 4 (RT 29.6 min) were quantified in similar amounts in systems at both test concentrations, whereas the amount of the major degradate designated as Polar Material (RT 2.8 min) was produced to a lesser extent at the 100 ug/L treatment level.

Test Systems Applied with the Test Item at 10 ug/L
The amount of Unknown 1 increased to a maximum mean of 17.4% applied radioactivity by Day 13, then declined to a mean of 0.5% applied radioactivity by Day 29. Unknown 1 then remained at a similarly low level up to Day 62. The amount of Unknown 3 increased to a maximum mean of 20.4% applied radioactivity by Day 29, then declined to a mean of 4.8% applied radioactivity by Day 62. The amount of Polar Material increased to a maximum mean of 23.3% applied radioactivity by Day 29, then declined to a mean of 17.4% applied radioactivity by Day 62. Up to 18 additional, unidentified minor degradation products were resolved and present at low levels such that further identification was not required.

Test Systems Applied with the Test Item at 100 µg/L
The amount of Unknown 1 increased to a maximum mean of 16.3% applied radioactivity by Day 29, then declined to a mean of 0.3% applied radioactivity by Day 43. Unknown 1 was detected at a higher level in one replicate after 62 days incubation; however, this result could be considered anomalous. The amount of Unknown 3 increased to a maximum mean of 27.0% applied radioactivity by Day 43, then declined to a mean of 21.6% applied radioactivity by Day 62. The amount of Unknown 4 increased to a maximum mean of 5.9% applied radioactivity by Day 29, then declined to a mean of 3.1% applied radioactivity by Day 62. The amount of Polar Material increased to a maximum mean of 7.8% applied radioactivity by Day 43. Polar Material was detected at a higher level in one replicate after 62 days incubation; however, this result could be considered anomalous. Up to 18 additional, unidentified minor degradation products were resolved and present at low levels such that further identification was not required.

Kinetic Analysis
The estimated DT50 values for the decline of Unknown 1 in the surface water were 3.9 days and 3.1 days at 10 ug/L and 100 ug/L concentrations, respectively.
The estimated DT50 values for the decline of Unknown 3 in the surface water were 23.8 days and 59.5 days at 10 ug/L and 100 ug/L concentrations, respectively; however, the kinetic data are limited to two time intervals for the 100 ug/L concentration.
The estimated DT50 value for the decline of Unknown 4 in the surface water was 38.9 days at the 100 ug/L concentration.
The estimated DT50 value for the decline of Polar Material in the surface water was 75.3 days at the 10 ug/L concentration.
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
Characterization of the Test System
The surface water was microbiologically active throughout the incubation period and there were no appreciable differences in levels of the various organisms at the end of the incubation period between untreated samples and samples treated with solvent. During the incubation period, oxygen levels in the water (94.1 to 104.6% air saturated value) were indicative of an aerobic surface water. The temperature of the room remained within the range 12 +/- 2 deg C throughout the incubation period.

Recovery and Distribution of Radioactivity
The total recoveries of radioactivity were 80.4 - 100.4% applied radioactivity. Samples with low total recoveries of radioactivity (<90%) tended to be those that were sampled in the latter stages of the study (≥29 days) at both concentration levels. Volatile radioactivity (14CO2) accounted for a maximum of 39.5% applied radioactivity. For some samples where the total recoveries of radioactivity were below the 90% target, acetonitrile washes of the test vessel and magnetic stir bar were performed and no significant amount of radioactivity was recovered. The total recoveries of radioactivity in the sterile surface water were 86.4 - 97.5% applied radioactivity. The mean amounts of parent material remaining in the sterile controls after 62 days in the surface water treated at 10 µg/L and 100 µg/L were 6.5% and 37.6% applied radioactivity, respectively. Volatile radioactivity (14CO2) accounted for a maximum of 2.8% applied radioactivity.

Chromatographic Analysis
HPLC analysis of the surface water resolved up to 22 components in addition to parent material, including four major degradates; several other minor degradates were distributed throughout the chromatograms but did not contain any discrete, resolved radioactive components. The amount of parent material in the surface water declined at a faster rate in the test systems treated with 10 ug/L. The mean amount of parent material in the test system treated at 10 ug/L (nominal) declined from 90.1% of applied radioactivity at the time of application to 1.6% after 62 days of incubation. The mean amount of parent material in the test system treated at 100 ug/L (nominal) declined from 96.2% of applied radioactivity at the time of application to 5.2% after 43 days of incubation. Parent material was detected at a higher level in one replicate after 62 days incubation; however, this result could be considered anomalous.

Identification of Radioactive Components
The identity of parent material applied was established by HPLC co-chromatographic correspondence with non-radiolabeled reference material by co-injection.

Transformation Pathway
A pathway for the transformation of the parent material in surface water will be proposed based upon the degradation identification by liquid chromatography with mass spectrometry (LC-MS).

Results with reference substance:
The total recoveries of radioactivity in the surface water treated with the reference material, [14C]-benzoic acid, at a concentration of 10 ug/L were 88.2 – 94.2% applied radioactivity after 14 days. Recoveries below the target of 90% applied radioactivity are attributed to incomplete trapping of CO2. Direct volatile radioactivity, all associated with 14CO2, accounted for 58.5 – 61.7% applied radioactivity after 14 days.

The test was shown to be valid as the reference control degraded within the expected time interval.
Validity criteria fulfilled:
yes
Conclusions:
The parent material degraded in aerobic surface water with estimated DT50 values of 10.3 days (10 ug/L applied) and 16.0 days (100 ug/L applied). The parent material degraded to Unknowns 1, 3 and 4 and Polar Material, accounting for mean maximum recoveries of 17.4, 27.0, 5.9 and 23.3% applied radioactivity, respectively. Identification of these major degradates is in progress.
Executive summary:

The biodegradation and fate of the test material was investigated at two concentrations in aerobic surface water under laboratory conditions according to OECD Testing Guideline No. 309. Surface water was treated with radiolabeled test material at nominal application rates of 10 ug/L and 100 ug/L. Treated surface water samples were attached to air flow lines with traps to collect carbon dioxide and incubated with continuous stirring to maintain aerobic conditions at 12 +/- 2 deg C in darkness for periods of up to 62 days. 

Total recoveries of radioactivity (mass balances) for samples treated at 10 ug/L and 100 ug/L were between 80.4 and 100.4% applied radioactivity. Samples with low total recoveries of radioactivity (<90%) tended to be those that were sampled in the latter stages of the study (≥29 days) at both concentration levels. Carbon dioxide accounted for a maximum of 39.5% applied radioactivity.

Separate sterile samples were treated to provide abiotic controls. Total recoveries of radioactivity (mass balances) for these sterile samples treated at 10 ug/L and 100 ug/L were between 86.4% and 97.5% applied radioactivity. Carbon dioxide accounted for a maximum of 2.8% applied radioactivity.

The mean amount of parent material in the test system treated at 10 ug/L (nominal) declined from 90.1% of applied radioactivity at the time of application to 1.6% after 62 days of incubation. The mean amount of parent material in the test system treated at 100 ug/L declined from 96.2% of applied radioactivity at the time of application to 5.2% after 43 days of incubation. Parent material was detected at a higher level in one replicate in the test system treated at 100 ug/L after 62 days incubation; however, this result could be considered anomalous.

The estimated DT50 values for the decline of parent material in aerobic surface water were 10.3 days and 16.0 days at 10 ug/L and 100 ug/L concentrations, respectively. The estimated DT90 values for the decline of parent material in aerobic surface water were 34.2 days and 53.2 days at 10 ug/L and 100 ug/L concentrations, respectively.

Analysis of the surface water by HPLC resolved up to 22 components in addition to parent material, including 4 major degradates; several other minor degradates were distributed throughout the chromatograms but did not contain any discrete, resolved radioactive components.  Analysis of the surface water samples showed that the applied parent material degraded to Unknown 1, Unknown 3, Unknown 4 and an unknown polar material, accounting for maximums of 21.1, 30.5, 7.7 and 25.4% applied radioactivity, respectively. Degradate identification is underway. Other (unidentified) degradation products were present individually at ≤5.8% applied radioactivity.

Description of key information

Surface Water Mineralization

The parent material degraded in aerobic surface water with estimated DT50 values of 10.3 days (10 ug/L applied) and 16.0 days (100 ug/L applied). The parent material degraded to Unknowns 1, 3 and 4 and Polar Material, accounting for mean maximum recoveries of 17.4, 27.0, 5.9 and 23.3% applied radioactivity, respectively.  Identification of these major degradates is in progress.

Aquatic Sediment Transformation

The parent material dissipated rapidly from the water of aquatic sediment systems incubated under aerobic conditions, with estimated DT50 values of 5.0 days (Calwich Abbey Lake) and 6.7 days (Emperor Lake).  Decline in the overall system incubated under aerobic conditions corresponded to estimated DT50 values of 240 days (Calwich Abbey Lake) and 215 days (Emperor Lake).  

The parent material dissipated slowly from the water of aquatic sediment systems incubated under anaerobic conditions, with estimated DT50 values of 46 days (Calwich Abbey Lake) and 39 days (Emperor Lake).  Decline in the overall system incubated under anaerobic conditions corresponded to estimated DT50 values of 958 days (Calwich Abbey Lake) and 1660 days (Emperor Lake).  

The parent material was degraded to one major degradate, Unknown 1 (up to a mean of 6.8% after 100 days) in the total system and 15 low level unidentified degradates (mean recovery did not exceed 2.9% applied radioactivity for any one minor degradate on any sampling occasion), was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide.

Key value for chemical safety assessment

Additional information

Surface Water Mineralisation

The biodegradation and fate of the test material was investigated at two concentrations in aerobic surface water under laboratory conditions according to OECD Testing Guideline No. 309. Surface water was treated with radiolabeled test material at nominal application rates of 10 ug/L and 100 ug/L. Treated surface water samples were attached to air flow lines with traps to collect carbon dioxide and incubated with continuous stirring to maintain aerobic conditions at 12 +/- 2 deg C in darkness for periods of up to 62 days. 

Total recoveries of radioactivity (mass balances) for samples treated at 10 ug/L and 100 ug/L were between 80.4 and 100.4% applied radioactivity. Samples with low total recoveries of radioactivity (<90%) tended to be those that were sampled in the latter stages of the study (≥29 days) at both concentration levels. Carbon dioxide accounted for a maximum of 39.5% applied radioactivity.

Separate sterile samples were treated to provide abiotic controls. Total recoveries of radioactivity (mass balances) for these sterile samples treated at 10 ug/L and 100 ug/L were between 86.4% and 97.5% applied radioactivity. Carbon dioxide accounted for a maximum of 2.8% applied radioactivity.

The mean amount of parent material in the test system treated at 10 ug/L (nominal) declined from 90.1% of applied radioactivity at the time of application to 1.6% after 62 days of incubation. The mean amount of parent material in the test system treated at 100 ug/L declined from 96.2% of applied radioactivity at the time of application to 5.2% after 43 days of incubation. Parent material was detected at a higher level in one replicate in the test system treated at 100 ug/L after 62 days incubation; however, this result could be considered anomalous.

The estimated DT50 values for the decline of parent material in aerobic surface water were 10.3 days and 16.0 days at 10 ug/L and 100 ug/L concentrations, respectively. The estimated DT90 values for the decline of parent material in aerobic surface water were 34.2 days and 53.2 days at 10 ug/L and 100 ug/L concentrations, respectively.

Analysis of the surface water by HPLC resolved up to 22 components in addition to parent material, including 4 major degradates; several other minor degradates were distributed throughout the chromatograms but did not contain any discrete, resolved radioactive components.  Analysis of the surface water samples showed that the applied parent material degraded to Unknown 1, Unknown 3, Unknown 4 and an unknown polar material, accounting for maximums of 21.1, 30.5, 7.7 and 25.4% applied radioactivity, respectively. Degradate identification is underway. Other (unidentified) degradation products were present individually at ≤5.8% applied radioactivity.

Aquatic Sediment Transformation

The fate of the test material was studied in two natural aquatic sediment systems under laboratory conditions according to OECD Guideline 308. The sediment from Emperor Lake was a sandy clay loam with an acidic pH and low organic carbon content, while that from Calwich Abbey Lake was a neutral silt loam with a higher organic carbon content. Samples of each aquatic sediment system were allowed to acclimatize separately under aerobic or anaerobic conditions before being treated with radiolabelled test material at a rate of 0.1 mg/L based on the amount of water in the test vessel including that present within the sediment. The samples were incubated under aerobic or anaerobic conditions at about 12 deg C in darkness for periods of up to 100 days.

 

Mean total recoveries of radioactivity (mass balances) for both aquatic sediments incubated under aerobic or anaerobic conditions were between 92.7% and 101.5% applied radioactivity. 

 

In Calwich Abbey Lake aquatic sediment incubated under aerobic conditions, the radioactivity in the water layer declined from a mean of 95.7% applied radioactivity at time zero to 3.7% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 91.7% applied radioactivity after 30 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sedimentaccounted for a mean of ≤7.3% applied radioactivitythroughout. Volatile radioactivity accounted for a maximum mean of 3.5% applied radioactivity after 100 days.

 

In Emperor Lake aquatic sediment incubated under aerobic conditions, the radioactivity in the water layer declined from a mean of 92.0% applied radioactivity at time zero to 4.0% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 89.3% applied radioactivity after 58 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤8.5% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 4.1% applied radioactivity after 100 days.

 

In Calwich Abbey Lake aquatic sediment incubated under anaerobic conditions, the radioactivity in the water layer declined from a mean of 90.3% applied radioactivity at time zero to 24.8% after 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 70.6% applied radioactivity after 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤5.6% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 1.2% applied radioactivity after 100 days.

 

In Emperor Lake aquatic sediment incubated under anaerobic conditions, the radioactivity in the water layer declined from a mean of 86.0% applied radioactivity at time zero to 21.6% after 57 days and remained at a similar level up to 100 days of incubation. In the sediment, the total radioactivity increased to a mean of 73.8% applied radioactivity after 57 days and remained at a similar level up to 100 days. The proportion of remaining non-extractable radioactivity in the sediment accounted for a mean of ≤7.4% applied radioactivity throughout. Volatile radioactivity accounted for a maximum mean of 1.9% applied radioactivity after 100 days.

 

DT50 and DT90 values were estimated using single first order kinetics modelling based on the observed degradation of the parent test material from the water, the sediment and from the total aquatic sediment system at approximately 12 deg C. In the two aerobic systems, DT50 values were determined to be 5.03 - 6.67 days (water), 226 - 406 days (sediment), and 215 - 240 days (whole system), and DT90 values ranged from 16.7 - 22.2 days (water), 749 - 1350 days (sediment), and 713 - 799 days (whole system). In the two anaerobic systems, DT50 values were determined to be 39 - 46.4 days (water), 1390 days (sediment), and 958 - 1660 days (whole system), and DT90 values ranged from 130 - 154 days (water), 4610 days (sediment), and 3180 - 5500 days (whole system). The DT values for sediment were estimated only for Emperor Lake only, as there was no decline in parent material in Calwich Abbey Lake sediments.

The test material was degraded to one major degradate, Unknown 1 (up to a mean of 6.8% in the total system) and 15 low level unidentified degradates (mean recovery did not exceed 2.9% applied radioactivity for any one minor degradate on any sampling occasion), was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide.