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Environmental fate & pathways

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:
04th July 2018 to 7th August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
[14C]-C22 ATQ
IUPAC Name:
[14C]-C22 ATQ
Test material form:
liquid
Remarks:
Dissolved in Ethanol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: Pharmaron-UK Ltd/CFQ43502

RADIOLABELLING INFORMATION

- Radiochemical purity: 99.8%

- Specific activity: 2.15 GBq/mmol

- Locations of the label: N,N,N-trimethyldocosan-1-aminium-14-14C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL


- Storage condition of test material: ca. -20°C

- Stability under test conditions: Stable

- Solubility and stability of the test substance in the solvent/vehicle: Soluble in ethanol vehicle

FORM AS APPLIED IN THE TEST (if different from that of starting material)

In Solution (Ethanol)

OTHER SPECIFICS:

Molecular weight: 406 g/mol

Molecular formula: CH3(CH2)21N(Cl)(CH3)3
Radiolabelling:
yes
Remarks:
14C labelled

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
Representative High Organic Carbon Water

- Location: Calwich Abbey, Calwich Abbey Lake, Calwich, Staffordshire, England,
52°59’6.4”N, 1°48’38.3”W (OSGB-SK127431)

- Sampling depth: 20-30 cm

- Contamination history: location known not to be in the catchment area of predominantly industrial or domestic effluent discharges or human activities and should not have been contaminated with DTAC or its structural analogues within the previous five years.

- Procedure: Scooped from lake by bucket

- Storage conditions: ca. 2-8°C

- Storage length: 9 days

- Temperature (°C) at time of collection: 23.6°C

- pH at time of collection: 8.50

- Electrical conductivity: 499 µS/cm

- Redox potential (mv) initial/final: 96.7/212.8

- Oxygen concentration (mg/l) initial/final: 8.14/8.20

- Total organic carbon (%): 5.59 mg/L

- Water filtered: yes

- Type and size of filter used, if any: 212 µm

Representative Low Organic Carbon Water

- Location: Lumsdale, Middle Pond Lumsdale, Derbyshire, England,
N 53° 8’ 39.2” N 1° 32’ 0.8”W / SK31298 60887

- Sampling depth: 40-50 cm


- Contamination history: location known not to be in the catchment area of predominantly industrial or domestic effluent discharges or human activities and should not have been contaminated with DTAC or its structural analogues within the previous five years.

- Procedure: Scooped from lake by bucket

- Storage conditions: ca. 2-8°C


- Storage length: 8 days

- Temperature (°C) at time of collection: 13.0°C

- pH at time of collection: 7.51

- Electrical conductivity: 154 µS/cm

- Redox potential (mv) initial/final: 57.2/252.7

- Oxygen concentration (mg/l) initial/final: 7.63/8.45

- Total organic carbon (%): 2.52 mg/L

- Water filtered: yes

- Type and size of filter used, if any: 212 µm
Details on source and properties of sediment:
- Details on collection (e.g. location, sampling depth, contamination history, procedure):

Representative High Organic Carbon Sediment

- Location: Calwich Abbey, Calwich Abbey Lake, Calwich, Staffordshire, England,
52°59’6.4”N, 1°48’38.3”W (OSGB-SK127431)

- Sampling depth: 1 m

- Contamination history: location known not to be in the catchment area of predominantly industrial or domestic effluent discharges or human activities and should not have been contaminated with DTAC or its structural analogues within the previous five years.

- Procedure: Scooped from the top 5 cm of the sediment onto bank to drain slightly

- Storage conditions: ca. 2-8°C

- Storage length: 9 days

- Textural classification (i.e. %sand/silt/clay): Silt Loam (USDA) Sand 12%/ Silt 69%/ Clay 19%

- Organic carbon (%): Initial: 5.2, final 5.5

- Redox potential (mv) initial/final: -342/-252

- Dry matter (%): Initial: 16.6, final: 23.6

- Biomass (%TOC): Initial: 5.32, final: 3.67

- Biomass (mg C/kg): Initial: 2765, final: 2016

- Sediment samples sieved: yes

- Type and size of filter used, if any: 2 mm

Representative Low Organic Carbon Sediment

- Location: Lumsdale, Middle Pond Lumsdale, Derbyshire, England,
N 53° 8’ 39.2” N 1° 32’ 0.8”W / SK31298 60887

- Sampling depth: 1 m

- Contamination history: location known not to be in the catchment area of predominantly industrial or domestic effluent discharges or human activities and should not have been contaminated with DTAC or its structural analogues within the previous five years.


- Procedure: Scooped from the top 5 cm of the sediment onto bank to drain slightly

- Storage conditions: ca. 2-8°C


- Storage length: 8 days

- Textural classification (i.e. %sand/silt/clay): Loamy sand (USDA) Sand 88%/ Silt 5%/ Clay 7%

- Organic carbon (%): Initial: 0.84, final 1.16

- Redox potential (mv) initial/final: -463/-418

- Dry matter (%): Initial: 51.4, final: 52.7

- Biomass (%TOC): Initial: 3.63, final: 2.47

- Biomass (mg C/kg): Initial: 305, final: 287

- Sediment samples sieved: yes

- Type and size of filter used, if any: 2 mm
Duration of test (contact time):
100 d
Initial test substance concentrationopen allclose all
Initial conc.:
99 µg/L
Based on:
test mat.
Remarks:
Low organic carbon sediment
Initial conc.:
103 µg/L
Based on:
test mat.
Remarks:
High organic carbon sediment
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
Details on study design:
TEST CONDITIONS

- Volume of test solution/treatment: 33 µL for High Carbon Sediment, 13 µL for Low Carbon Sediment

- Solubilising agent (type and concentration if used): Ethanol

- Test temperature: 12 ± 2°C

- pH: High Carbon Sediment: 8.11, Low Carbon sediment: 6.82

- pH adjusted: no

- Aeration of dilution water: Continuous

- Continuous darkness: yes

- Any indication of the test material adsorbing to the walls of the test apparatus: yes, extracted where possible

- Other: Silanization of glassware investigated due to adsorbance of test material to glassware found not be not effective then not used

TEST SYSTEM

- Culturing apparatus: Glass flask, air bubbled through system, connections made with glass and neoprene tubing

- Number of culture flasks/concentration: High Carbon Sediment: 14 flasks/103 µg/L, Low Carbon Sediment: 14 flasks/99 µg/L

- Method used to control oxygen conditions: air continuously bubbled through system

- Measuring equipment: Portable probes for pH, dissolved oxygen and redox potential of water and sediment

- Test performed in open system: With continuous flow of air


- Details of trap for CO2 and volatile organics if used: Duplicate 2M sodium hydroxide traps for CO2, single ethylene glycol trap for volatiles attached to each test flask under negative pressure

SAMPLING


- Sampling frequency: 0, 3, 7, 14, 28 and 100 days, volatile traps had further changes on day 21 and 60

- Sampling method used per analysis type: Whole test flask removed at each sampling point. Water was separated from sediment by decanting into separate vessel containing 30 mL of methanol.

Sediment transferred using 100 mL of 5 mM ammonium formate in methanol with 0.1% formic acid then separated with centrifugation. Extraction repeated a second time. Third extraction using methanol only was used.

Sediment residues air-dried prior to combustion of subsamples.

Volatile traps were removed at each sampling interval and replace with fresh media where required.

- Sample storage before analysis: Study samples were analysed as soon as possible after generation (typically on the day of sampling or stored under refrigeration (ca. 4 - 8°C) if required the next day).


STATISTICAL METHODS:

The dissipation (DT50/90 (water)) in the water phase of the test item and disappearance from the whole test system (DT50/90 (total)) were calculated using a single first-order kinetic model

Results and discussion

Test performance:
The overall mass balance was low throughout the study for the high organic carbon water-sediment with a mean of 86.90% (range 80.37 - 95.69%) of applied radioactivity (AR) recovered. The low mass balance was extensively investigated and was determined that the causes were likely the result of poor water solubility and binding effects of the test item to all vessel surfaces upon transfer. As throughout the study only test item was observed with no significant metabolites detected. The experimenters’ are therefore confident that the losses observed are of test item only and thus the low mass balance did not significantly affect the integrity of the study.
Mean total recoveryopen allclose all
Compartment:
natural water / sediment: freshwater
% Recovery:
86.9
St. dev.:
4.65
Remarks on result:
other: High organic carbon sediment
Compartment:
natural water / sediment: freshwater
% Recovery:
93.66
St. dev.:
4.92
Remarks on result:
other: Low organic sediment
Half-life of parent compound / 50% disappearance time (DT50)open allclose all
Key result
Compartment:
natural water / sediment: freshwater
DT50:
29 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: High organic carbon sediment
Key result
Compartment:
natural water / sediment: freshwater
DT50:
56.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Low organic carbon sediment
Other kinetic parameters:
first order rate constant
Transformation products:
no
Details on transformation products:
A single component (R1) was detected in the Calwich Abbey sediment extract at <1% AR at day 100. In Lumsdale total system no transformation products were detected as detailed by the study objectives.
Only a single degradation product (R1) was detected and did not warrant further structural investigation as <5% AR; however the major component assigned as DTAC was confirmed in selected samples by TLC analysis using an authentic reference standard of non-labelled DTAC. The results of the TLC analysis for all samples co-spotted with DTAC reference standard showed a single major spot consistent with the Rf for the DTAC reference standard. Confirming that [14C]DTAC was the major component present in the samples.
A single transformation product at <1% was detected in a single replicate of the day 100 Calwich Abbey sediment extract during the study. Since substantial levels of mineralization were observed it is hypothesized that the test item comprises a good substrate for microbial degradation, resulting in direct metabolism of the test item to carbon dioxide. Accordingly Docosyltrimethylammonium chloride is unlikely to persist for lengthy periods in the environment.
Details on results:
TEST CONDITIONS

- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes

- Anomalies or problems encountered (if yes):

For the Calwich Abbey water-sediment system the mass balance was generally below the targeted range (90-110%); however individual unit mass balances were all >80% AR. The low mass balance in the Calwich Abbey sediment was extensively investigated and the cause was likely the result of poor water solubility and binding effects of the test item to all vessel surfaces upon transfer. For this reason the lower than targeted mass balances have been accepted.

MAJOR TRANSFORMATION PRODUCTS

No major transformation products detected


MINOR TRANSFORMATION PRODUCTS

No minor transformation products detected

EXTRACTABLE RESIDUES

Calwich Abbey- high organic carbon

- % of applied amount at day 0: 9.04

- % of applied amount at end of study period: 25.03

NON-EXTRACTABLE RESIDUES

- % of applied amount at day 0: 7.79

- % of applied amount at end of study period: 29.95

MINERALISATION

- % of applied radioactivity present as CO2 at end of study: 29.67

VOLATILIZATION

- % of the applied radioactivity present as volatile organics at end of study: None

EXTRACTABLE RESIDUES

Lumsdale- low organic carbon

- % of applied amount at day 0: 17.38

- % of applied amount at end of study period: 29.11

NON-EXTRACTABLE RESIDUES

- % of applied amount at day 0: 6.79

- % of applied amount at end of study period: 20.62

MINERALISATION

- % of applied radioactivity present as CO2 at end of study: 41.14

VOLATILIZATION

- % of the applied radioactivity present as volatile organics at end of study: None

Applicant's summary and conclusion

Validity criteria
Validity criteria fulfilled:
yes
Conclusions:
Docosyltrimethylammonium Chloride (DTAC) was steadily degraded in two water-sediment systems to carbon dioxide. Dissipation rates of DTAC from the water phases in the Calwich Abbey and Lumsdale systems, under the experimental conditions gave DT50 (water) values of 4.16 days and 1.19 days, respectively. Disappearance times calculated for DTAC in the total system under the experimental conditions gave DT50 (total) values of 29.0 days and 56.7 days obtained for Calwich Abbey and Lumsdale sediment systems, respectively.
Executive summary:

Study Design

The rate of disappearance of 14C-labelled Docosyltrimethylammonium chloride ([14C]DTAC) was investigated in two different water-sediment systems: Calwich Abbey, a representative high organic carbon sediment and Lumsdale, a representative low organic carbon sediment.  [14C]DTAC was applied at a nominal application rate of 100 µg/L to the water phase.  Each sediment type was maintained under aerobic conditions and incubated in the dark at ca. 12 ± 2°C for 100 days.  Duplicate samples were taken for analysis at six intervals (0, 3, 7, 14, 28 and 100 days).  Microbial biomass samples were similarly incubated with separate initial and final biomass samples and analysed using the fumigation-extraction method.

At each sampling interval, complete test systems were removed in duplicate.  The water phase was separated from the sediment into another vessel.  The sediment phase was extracted four times with 5 mM ammonium formate in methanol with 0.1% formic acid and twice with methanol.  Any volatile radioactivity was continuously flushed from the test vessels and collected in trapping solutions and analysed.  Sediment residues after extraction were air dried and aliquots combusted to determine the radioactivity content.  A mass balance was determined for each sample.

Samples containing >5% applied radioactivity (AR) were analysed by high performance liquid chromatography (HPLC).

Results

The achieved application rates were as follows:

Calwich Abbey: 103 µg/L               Repeat vessels:                116 µg/L

Lumsdale: 99 µg/L                            Repeat vessels:`               114 µg/L

A summary of the mass balance data for the aerobic incubation is as follows:

Water Residues

Water residues declined with time in all test systems.

 

Total water residues at zero days

Calwich Abbey: Mean 74.54%
Lumsdale: Mean 75.44%

Total water residues at end of study

Calwich Abbey: Mean 1.00%
Lumsdale: Mean 0.53%

Extractable Residues

Extractable residues increased with time in all test systems.

 

Total extractable residues at zero days

Calwich Abbey: Mean 9.04%
Lumsdale: Mean 17.38%

Total extractable residues at end of study

Calwich Abbey: Mean 25.03%
Lumsdale: Mean 29.11%

Bound Residues

Bound residues at end of study (100 days)

Calwich Abbey: Mean 29.95%
Lumsdale: Mean 20.62%

Carbon Dioxide

Significant amounts of radioactivity were evolved as carbon dioxide throughout the course of the study.

Carbon dioxide evolved at end of study

Calwich Abbey: Mean 29.67%
Lumsdale: Mean 41.14%

Organic Volatiles

No organic volatiles were detected

Total Radioactivity

Sum of activity in the surface water, sediment extracts, sediment residue on combustion and that trapped as carbon dioxide in the 2 M sodium hydroxide traps and organic volatiles in ethylene glycol.

Mean Recovery at Zero Days

91.37% (Calwich Abbey) and 99.61% (Lumsdale) of applied dose

Overall Recovery (all samples)

Range 80.37 to 102.42% of applied radioactivity

Calwich Abbey: Mean 86.90%
Lumsdale: Mean 93.66%

       

For the Calwich Abbey water-sediment system the mass balance was generally below the targeted range (90-110%); however individual unit mass balances were all >80% AR.  The low mass balance in the Calwich Abbey sediment was extensively investigated and the cause was likely the result of poor water solubility and binding effects of the test item to all vessel surfaces upon transfer.  For this reason the lower than targeted mass balances have been accepted.

Radioactivity was rapidly transferred from the water to the sediment phase and vessel surfaces.  The radioactivity in the water declined to a mean of 1.00% AR and 0.53% AR in the Calwich Abbey and Lumsdale water sediment systems respectively by the end of the study.

Significant mineralization was observed up to a mean of between 29.67 - 41.14% carbon dioxide evolved by the end of the study for Calwich Abbey and Lumsdale respectively.  No organic volatiles were detected.  Since substantial levels of mineralization were observed during this study it is hypothesized that the test item comprises a good substrate for microbial degradation, resulting in direct metabolism of the test item to carbon dioxide.  Accordingly DTAC is unlikely to persist for lengthy periods in the environment.

Under aerobic conditions, the dissipation rates of DTAC from the water phases in the Calwich Abbey and Lumsdale systems, under the experimental conditions gave DT50 (water) values of 4.16 and 1.19 days and DT90 (water) values of 13.8 and 3.94 days using SFO model, respectively.  Disappearance times calculated for DTAC in the total system under the experimental conditions gave DT50 (total) values of 29.0 and 56.7 days and DT90 (total) values of 96.4 and >100 days obtained for Calwich Abbey and Lumsdale sediment systems, respectively 

DTAC was confirmed to be present in all water samples and sediment extracts analysed by HPLC with secondary confirmation using thin layer chromatography (TLC).

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