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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
jan 14-march 19, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Operational parameters of the CAS were sometimes out of recommended range (dissolved oxygen concentration, mixed liquor suspended solids). Because this test is operated using environmental samples, these parameters can not always be maintained within prescribed limits. These fluctuations would not be expected to have a major impact on the test results.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): diethyl ester dimethyl ammonium chloride (E 4607.01)
- Substance type: pure act. ingr. substance
- Physical state: liquid, A 5 % dispersion (E 4607.01) of DEEDMAC was prepared for dosing the test system using the parent test chemical E 4429.01.
- Preparation of Dispersion solution : 5% aqueous dispersion of DEEDMAC made up in 3.7% HCl Solution (pH 2.6)
- Stability under test conditions: yes
- Storage condition of test material: rt

Radiolabelling:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of Activated Sludge: Bocholt Wastewater treatment plant that receives primarily domestic wastewater
- Storage conditions: RT
- Initial cell/biomass concentration: 3.44 g/L in activated sludge
- Source of nutrient solution (wastewater treatment plant influent): Wastewater from Bocholt wastewater treatment plant, fresh wastewater was brought in 2x per week over the course of experiment.


Duration of test (contact time):
6 wk
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Test temperature: 17.7-20.9 C
- pH: 6.99-8.56
- pH adjusted: no
- Suspended solids concentration: 1.34 - 9.3 mg/L
- Any indication of the test material adsorbing to the walls of the test apparatus: not measured
- Other: dissolved oxygen in aeration basin range 6.8-8.8 mg/L


TEST SYSTEM
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: aeration of the aeration vessel
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: none used


SAMPLING
- Sampling frequency: 5 times per week
- Sampling method:
100 ml of dosing solution (pH 2.5) stored refrigerated for maximum of 7 days
2x 500 ml effluent in polypoplyene containing 3% formaldehyde store refrigerated for max of 7 days
sludge solids 2x 100 ml of mlss add 3% formaldehyde. Centrifuge and decant supernatant layer, dry sludge solids under nitrogen.


CONTROL AND BLANK SYSTEM
- Inoculum blank: YES


:
Reference substance:
not required
Test performance:
Operational parameters of the CAS were sometimes out of recommended range (dissolved oxygen concentration, mixed liquor suspended solids). Because this test is operated using environmental samples, these parameters can not always be maintained within prescribed limits. These flucutations would not be expected to have a major impact on the test results.
% Degr.:
> 99
Parameter:
test mat. analysis
Sampling time:
3 wk
Remarks on result:
other: average value during the three weeks removal period
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
Removal of reaction products of MDEA-Esterquat (C16-18/C18 unsaturated fatty acid with methyl diethanolamine, MeCl quaternized) exceeded 99.7 %. This was based on average measured effluent concentrations of 7.067 +/- 4.788 µg/L. However, the detection limit for the effluent samples was 12.72 µg/L. Based on the fluctuation in the effluent concentration and the highest effluent concentration observed (16 µg/L) the minimal removal during the test phase would have been 99.1%. The concentration of MDEA-Esterquat on the activated sludge solids was 50.43 µg/g over the three week removal period. Mass balance showed removal to be mainly due to biodegradation (99.5%).


The removal of MDEA-Esterquat in an aerobic sewage treatment simulation test was investigated in a continuous activated sludge test system using influent and activated sludge collected from the wastewater treatment plant at Bochum (Germany) that receives primarily domestic wastewater. The study was conducted in accordance with OECD 303A, Simulation test - Aerobic Sewage Treatment, Activated Sludge Units. Two OECD Confirmatory Test Units were operated in parallel, one dosed at 2 mg/L MDEA-Esterquat concentration and the second had no test chemical (blank unit). The dosing solution and activated sludge solids were analyzed by dilution with a solvent mixture (chloroform/acetonitrile 80/20) and adjusting solution to pH 2.5 with acetic acid and addition of deuterated MDEA-Esterquat. Samples of effluent were analyzed directly after addition of deuterated MDEA-Esterquat (separation method: flow injection mass spectroscopy (FI/MS)).The average measured effluent concentration was 7.067 +/- 4.788 µg/L. However, the detection limit for the effluent samples was 12.72 µg/L. Based on the fluctuation in the effluent concentration and the highest effluent concentration observed (16 ug/L) the minimal removal during the test phase would have been 99.1%. The concentration of MDEA-Esterquat on the activated sludge solids was 50.43 µg/g over the three week removal period. The mass balance calculated in the 3 weeks removal period showed that more than 99% of the removed MDEA-Esterquat was eliminated by primary biodegradation.

Validity criteria fulfilled:
yes
Conclusions:
The removal of MDEA-Esterquat C16-18 and C18 unsatd. in an aerobic sewage treatment simulation test was investigated in a continuous activated sludge test system using influent and activated sludge collected from the wastewater treatment plant at Bochum (Germany) that receives primarily domestic wastewater. The study was conducted in accordance with OECD 303A, Simulation test - Aerobic Sewage Treatment, Activated Sludge Units and GLP standards. On average >99% parent MDEA-Esterquat C16-18 and C18 unsatd. was removed during the three weeks removal period.
Executive summary:

The removal of MDEA-Esterquat C16-18 and C18 unsatd. in an aerobic sewage treatment simulation test was investigated in a continuous activated sludge test system using influent and activated sludge collected from the wastewater treatment plant at Bochum (Germany) that receives primarily domestic wastewater. The study was conducted in accordance with OECD 303A, Simulation test - Aerobic Sewage Treatment, Activated Sludge Units and GLP standards. On average >99% parent MDEA-Esterquat was removed during the three weeks removal period. This was based on an average measured effluent concentration of 7.067 +/- 4.788 µg/L. However, the detection limit for the effluent samples was 12.72 µg/L. Based on the fluctuation in the effluent concentration and the highest effluent concentration observed (16 µg/L) the minimal removal during the test phase would have been 99.1%. The concentration of MDEA-Esterquat C16-18 and C18 unsatd. on the activated sludge solids was 50.43 µg/g over the three weeks removal period. The mass balance calculated in the 3 weeks removal period showed that more than 99% of the removed MDEA-Esterquat C16-18 and C18 unsatd. was eliminated by primary biodegradation.

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2, 1993 - November 30, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 314 - Accepted as a new guideline in 2008, is currently in the process of being adopted
Deviations:
yes
Remarks:
Early version of the newly adopted OECD 314 TG, "Simulation tests to assess the biodegradability of chemicals discharged in wastewater".
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): SS0197.01R, BFA and FV Base, N-Bis (talloyloxyethyl) - N- dimethyl ammonium chloride
- Substance type: SS0197.01R was a dispersion prepared from SS0132.01R, [N-14C CH3] -N-Bis (talloyloxyethyl) - N-
Dimethylammonium chloride and SS0134.01, N-Bis (talloyloxyethyl) - N-Dimethylammonium chloride.


SS0197.01R:
- Physical state: 125 ppm dispersion solution, lt blue in color, pH 2.5
- Expiration date of radiochemical substance (if radiolabelling): not given
- Stability under test conditions: Stabile based on Rad-TLC analysis of experiment
- Storage condition of test material: Ambient, dry storage

SS0132.01R:
- Physical state: solution, white,
- Expiration date of radiochemical substance (if radiolabelling): 6-1-93
- Storage condition of test material: Refrigerated, dry storage

SS0134.01
- Physical state: solid
- Stability under test conditions: not available
- Storage condition of test material: Refrigerated, dry storage
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge collected from Sycamore
Sewage Treatment Plant aeration basin, plant receives primarily domestic wastewater.

- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: Test conducted at room temperature ~22C
- Storage length: sludge stored one day prior to test intitation
- Preparation of inoculum for exposure: aerated until used
- Pretreatment: none
- Concentration of sludge: Activated sludge solids (MLSS) were adjusted to 2500 mg/L
- Initial cell/biomass concentration: unknown
Duration of test (contact time):
7 d
Initial conc.:
0.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
Details on study design:
One liter volumes of abiotic and biotic activated sludge (2500 mg/L) were dosed with 0.5 mg/L of test material and incubated for a seven day test period. The headspace of the flasks was purged with CO2 free air and the effluent gas from each flask was passed through a series of base traps to
capture carbon dioxide. The base traps were sampled and analyzed by LSC. Separate samples of activated sludge were removed from the test flasks to determine the amount of dissolved 14CO2 and to characterize the radioactivity remaining in the test system. Dissolved CO2 was determined by placing 10 ml aliquots of activated sludge in a sealed vial containing a wick soaked with 0.2 ml of 1.5 N KOH. After 24 hr, the wick was recovered and placed in UG cocktail and analyzed by LSC. Two 10 ml aliquots of sludge (one acidified from dissolved CO2 measurement, one not acidified) were flash frozen and freeze dried. The dried solids were extracted with methanol to recover parent and hydrolysis products. The methanol extracts were driedunder nitrogen and analyzed by Rad-TLC. The extracted solids were combusted to determine the amount of radioactivity associated with the sludge biomass. The test was conducted over a 7 day period. A complete mass balance is calculated for the 14C dosed to the test system.
Reference substance:
not required
% Degr.:
64.3
Parameter:
CO2 evolution
Sampling time:
7 d
% Degr.:
62.8
Parameter:
CO2 evolution
Sampling time:
7 d
% Degr.:
> 80
Parameter:
test mat. analysis
Sampling time:
24 h
Remarks on result:
other: based on LSC and Rad-TLC analysis
Compartment:
other: activated sludge
DT50:
>= 7 - <= 14 h
Type:
other: first order
Remarks on result:
other: first order model using nonlinear regression method
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
The two transformation products are known hydrolysis products of BFA base: HP-1 - mono fatty acid ester. HP-2 - diethanol dimethyl ammonium chloride.


- Formation and decline of each transformation product during test: The level of HP-2 increased from 0 to <20% in 24 hr and decreased to <5 % of the initial radioactivity afer day 7. HP-1 had virtually disappeared after 24 hr.
- Pathways for transformation: BFA base appears to be initially hydrolyzed to HP-1 which is rapidly hydrolyzed to HP-2 which is incorporated into biomass or evolved 14CO2 without further accumulation of intermediates. Hp-1 and HP-2 were both transient intermediates and did not accumulate.
- Other:
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Aerobicity was maintained using a stir plate and aeration with CO2 free air.

- Anomalies or problems encountered (if yes): No


MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: HP2 reached a maximum of 16% after 24 hr.
- Range of maximum concentrations in % of the applied amount at end of study period: HP2 was < 5% of applied radioactivity after day 7


TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: none


EXTRACTABLE RESIDUES
- % of applied amount at day 0: ~86%
- % of applied amount at end of study period: ~10%


NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:~ 20%
- % of applied amount at end of study period: ~20%


MINERALISATION
- % of applied radioactivity present as CO2 at end of study: ~65%


VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: not measured


STERILE TREATMENTS (if used) - Not applicable
- Transformation of the parent compound:
- Formation of transformation products:
- Formation of extractable and non-extractable residues:
- Volatilization:


RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):

The half life for parent based upon the first order rate (k1) of primary degradation equaled 7 to 14 hrs using the equation t1/2= 0.693/k1 (Larson 1979, 1981a). Based on the kinetics of mineralization to 14CO2, the half life for mineralization equaled 18 -24 hr

Validity criteria fulfilled:
not applicable
Conclusions:
The results of this experiment (in accordance with an early version of the newly adopted OECD 314) with non-adapted activated sludge indicated that MDEA-Esterquat C16-18 and C18 unsatd. and its hydrolysis products were fully biodegraded by microbes in unacclimated activated sludge. After 24 hr 20% of the parent remained, HP-1 (mono fatty acid ester) had virtually disappeared and HP-2 (diethanol dimethyl ammonium chloride) reached a maximum of 16%. The amount of activity incorporated into biomass was 25% and the amount trapped as 14CO2 was 35% during the same period. After seven days, the percentage of radioactivity converted to 14CO2 and incorporated into biomass totaled more than 75%, while the level of HP-2 remained at less than 5%. Based upon the kinetics of parent disappearance and metabolite production, it appears that MDEA-Esterquat C16-18 and C18 unsatd. is initially biologically hydrolyzed to HP-1, which very rapidly is hydrolyzed to HP-2, which is incorporated into biomass or evolved as 14CO2 without further accumulation of intermediates. HP-1 and HP-2 were both transient intermediates that did not accumulate. The half life for parent based upon the first order rate (k1) of primary degradation equaled 7 to 14 hrs using the equation t1/2=0.693/k1. Based on the kinetics of mineralization to 14CO2, the half life for mineralization equaled 18 -24 hr .
Executive summary:

The results of this experiment (in accordance with an early version of the newly adopted OECD 314) with non-adapted activated sludge indicated that 14C-MDEA-Esterquat C16-18 and C18 unsatd. (N-methyl group radiolabelled) and its hydrolysis products were fully biodegraded by microbes in unacclimated activated sludge. After 24 hr 20% of the parent remained, HP-1 (mono fatty acid ester) had virtually disappeared and HP-2 (diethanol dimethyl ammonium chloride) reached a maximum of 16%. The amount of activity incorporated into biomass was 25% and the amount trapped as 14CO2 was 35% during the same period. After seven days, the percentage of radioactivity converted to 14CO2 and incorporated into biomass totaled more than 75%, while the level of HP-2 remained at less than 5%. Based upon the kinetics of parent disappearance and metabolite production, it appears that MDEA-Esterquat C16-18 and C18 unsatd. is initially biologically hydrolyzed to HP-1, which very rapidly is hydrolyzed to HP-2, which is incorporated into biomass or evolved as 14CO2 without further accumulation of intermediates. HP-1 and HP-2 were both transient intermediates that did not accumulate. The half life for parent based upon the first order rate (k1) of primary degradation equaled 7 to 14 h using the equation t1/2=0.693/k1. Based on the kinetics of mineralization to 14CO2, the half life for mineralization equaled 18 -24 hr .

Description of key information

water: >99% removal on average during the 3 weeks removal period
sediment: not required

Key value for chemical safety assessment

Additional information

Simulation tests

Water

The removal of MDEA-Esterquat C16-18 and C18 unsatd. in an aerobic sewage treatment simulation test was investigated in a continuous activated sludge test system in accordance with OECD 303A, Simulation test - Aerobic Sewage Treatment, Activated Sludge Units and GLP standards.

Sediment

According to REACH regulation (Annex IX, 9.2.1.4, column II), the biodegradation simulation test in sediment does not need to be conducted if the substance is readily biodegradable.

The removal of MDEA-Esterquat C16-18 and C18 unsatd. in an aerobic sewage treatment simulation test was investigated in a continuous activated sludge test system using influent and activated sludge collected from the wastewater treatment plant at Bochum (Germany) that receives primarily domestic wastewater. The study was conducted in accordance with OECD 303A, Simulation test - Aerobic Sewage Treatment, Activated Sludge Units and GLP standards. On average >99% parent MDEA-Esterquat C16-18 and C18 unsatd. was removed during the three weeks removal period. This was based on an average measured effluent concentration of 7.067 +/- 4.788 µg/L. However, the detection limit for the effluent samples was 12.72 µg/L. Based on the fluctuation in the effluent concentration and the highest effluent concentration observed (16 ug/L) the minimal removal during the test phase would have been 99.1%. The concentration of MDEA-Esterquat C16-18 and C18 unsatd.  on the activated sludge solids was 50.43 µg/g over the three weeks removal period. The mass balance calculated in the 3 weeks removal period showed that more than 99% of the removed MDEA-Esterquat was eliminated by primary biodegradation.

The results of this experiment (in accordance with an early version of the newly adopted OECD 314) with non-adapted activated sludge indicated that 14C-MDEA-EsterquatC16-18 and C18 unsatd. (N-methyl group radiolabelled) and its hydrolysis products were fully biodegraded by microbes in unacclimated activated sludge. After 24 hr 20% of the parent remained, HP-1 (mono fatty acid ester) had virtually disappeared and HP-2 (diethanol dimethyl ammonium chloride) reached a maximum of 16%. The amount of activity incorporated into biomass was 25% and the amount trapped as 14CO2 was 35% during the same period. After seven days, the percentage of radioactivity converted to 14CO2 and incorporated into biomass totaled more than 75%, while the level of HP-2 remained at less than 5%. Based upon the kinetics of parent disappearance and metabolite production, it appears that MDEA-EsterquatC16-18 and C18 unsatd. is initially biologically hydrolyzed to HP-1, which very rapidly is hydrolyzed to HP-2, which is incorporated into biomass or evolved as 14CO2 without further accumulation of intermediates. HP-1 and HP-2 were both transient intermediates that did not accumulate. The half life for parent based upon the first order rate (k1) of primary degradation equaled 7 to 14 h using the equation t1/2=0.693/k1. Based on the kinetics of mineralization to 14CO2, the half life for mineralization equaled 18 -24 hr .

In an influent die-away experiment, cold and radiolabelled MDEA-Esterquat C16-18 and C18 unsatd.  were dosed at a combined concentration of 1 mg/L in raw domestic sewage. No mineralization was observed in the test system. Primary degradation was observed in the biotic treatment. The Rad-TLC analysis of the biotic treatment indicates that parent underwent degradation after a short lag period. The % parent remaining after 6, 24 and 48 h was 57%, 29% and 12%, respectively. Based on an initial recovery of ~70% from both treatments, it appears the half-life of parent in influent would be ~20 hr. Due to the very low recovery of radioactivity from the water extract after the 6 hr sampling interval and post lyophilization, the characterization of these extracts is inconclusive. However, it can be concluded that intermedates more polar than parent were present in the 24 and 48 samples because the majoirty of the radioactivity partitioned into the water phase and not the ethyl acetate phase. The study is not reliable due to methodological deficiencies. It can, however, be regarded as supportive study.