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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 314
Deviations:
no
GLP compliance:
no
Remarks:
The study was not originally conducted for REACH purposes.
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment
Details on source and properties of sediment:
Sediment was collected from the Lytle Creek located in Wilmington, Ohio. Surficial (top 2-3 cm) sediment and overlying water was collected using a plastic scoop at a site immediately below the Wilmington Wastewater Treatment Plant outfall. The plant services a population of about 17,000 and receives about 90% of its waste from domestic sources. The sediment was placed in plastic jars and kept on ice during transport and then stored in a 4°C cold room prior to the test. The sediment was characterized by the University of Wisconsin, Madison Soil & Plant Analysis Laboratory. The sediment type was sandy comprised of 95% sand, 2% silt and 3% clay. Organic content was 2.06%, total nitrogen content was 0.02%, and pH was 7.9.
Duration of test (contact time):
148 d
Initial conc.:
1.5 other: mg/kg dry weight
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
other: Metabolites
Details on study design:
Overview of experimental design
The radiolabeled test substance was incubated with biotic and abiotic sediment samples under static conditions. The sediment for the abiotic treatment was autoclaved for 90 min and amended with mercuric chloride at 1 g/L to inhibit microbial activity. The test systems consisted of replicate 1 mL samples of sediment with 0.1 mL overlying water in test tubes, which were individually dosed with the test substance at a final added concentration of 1.5 mg/kg dry weight.

Four replicate samples for the biotic treatment were prepared per sampling interval and all the biotic samples were incubated together in a sealed dessicator, which contained a 50 mL beaker containing 20 mL of 1.5 N KOH to trap any evolved 14CO2 in the headspace. In addition, the dessicator was continuously purged with CO2 -free air to maintain aerobic conditions, and the effluent gas was passed through a gas trapping system consisting of one empty trap followed by three base traps containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap.

At each sampling, levels of evolved and dissolved 14CO2 were determined for the biotic treatments. In addition, replicates of abiotic and biotic sediment were analyzed for parent, metabolites and non-extractable radioactivity. (Refer figures 1 and 2 at pages 9 and 10 of the study report (WTDS#68316) for diagrams of the test system and the analysis scheme).

TEST CONDITIONS
- Volume of test solution/treatment: 1 mL sediment with 0.1 mL overlying water
- Composition of medium: natural sediment, overlying water
- Additional substrate: no
- Solubilising agent: not used
- Test temperature: 22°C
- pH: not specified in the study report
- pH adjusted: no
- Aeration of dilution water: N/A
- Suspended solids concentration: N/A
- Continuous darkness: not specified in the study report
- Any indication of the test material adsorbing to the walls of the test apparatus: no

TEST SYSTEM
- Culturing apparatus: test tubes
- Number of culture flasks/concentration: 4 at each time point (biotic) and 2 at each point (abiotic)
- Method used to create aerobic conditions: CO2-free air continuously pumped through test system
- Measuring equipment: As per the details described in analytical methods.
- Test performed in closed vessels: yes (CO2-free air purged through system to measure evolved 14CO2 in traps)
- Details of trap for CO2: Details could be referred as per the overview of experimental design above.

SAMPLING
- Sampling frequency: Biotic treatment was sampled after 15 min and after 1, 2, 3, 6, 9, 14, 33, 61, and 148 days. The abiotic treatment was sampled less frequently.
- Sampling method: At each sampling, the dessicator was opened to change the internal base trap and to recover sufficient samples for dissolved 14CO2 and for characterizing residual radioactivity.
- Sample storage before analysis: not specified in study report


STATISTICAL METHODS: The parent loss and mineralization data were fit to a variety of first order decay and production equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D software (version 4.01).
Compartment:
biologically active treatment at end of test
% Recovery:
101
St. dev.:
0.3
Compartment:
abiotic control measured at end of test
% Recovery:
110.6
St. dev.:
2.1
Key result
% Degr.:
60.8
St. dev.:
0.3
Parameter:
CO2 evolution
Sampling time:
148 d
Remarks on result:
other: Mean of biotic flasks; aerobic
Key result
% Degr.:
14.4
St. dev.:
0.7
Parameter:
other: associated with solids
Sampling time:
148 d
Remarks on result:
other: Mean of biotic flasks; aerobic
Key result
% Degr.:
1.4
St. dev.:
0.6
Parameter:
other: metabolites
Sampling time:
148 d
Remarks on result:
other: Mean of biotic flasks; aerobic
Key result
% Degr.:
24.5
St. dev.:
0.05
Parameter:
other: parent
Sampling time:
148 d
Remarks on result:
other: Mean of biotic flasks; aerobic
Key result
% Degr.:
107.5
St. dev.:
3.7
Parameter:
other: parent
Sampling time:
148 d
Remarks on result:
other: Mean of abiotic flasks; aerobic
Key result
% Degr.:
3
St. dev.:
1.3
Parameter:
other: associated with solids
Sampling time:
148 d
Remarks on result:
other: : Mean of abiotic flasks; aerobic
Key result
Compartment:
sediment
DT50:
0.4 d
St. dev.:
0.1
Type:
other: two compartment first order model
Remarks on result:
other: Primary biodegradation; aerobic; compartment 1
Key result
Compartment:
sediment
DT50:
99 d
St. dev.:
4.2
Type:
other: two compartment first order model
Remarks on result:
other: Primary biodegradation; aerobic; compartment 2
Key result
Compartment:
sediment
DT50:
11.6 d
St. dev.:
1.5
Type:
other: first order
Remarks on result:
other: The rate of mineralization was determined using a first order model
Mineralization rate (in CO2):
0.006 d-1
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
Details on transformation products:
One metabolite was identified at Rf 0.57 which reached a maximum of 5.7% and subsequently disappeared.
Evaporation of parent compound:
no
Volatile metabolites:
not measured
Residues:
yes
Details on results:
Primary degradation: It was best described by a two compartment first order model (r2 ≥ 0.99). The process was biphasic with two pools (compartments) of material exhibiting different degradation rates. Pool A (compartment 1) presumably was readily bioavailable test material, in the aqueous phase. Pool B (compartment 2) presumably was less bioavailable test material, bound to solids (sorbed).

Primary degradation in pool A (compartment 1): 42.2%
Primary degradation in pool B (compartment 2): 64.8%

Mineralization (14CO2 production): It was best described by a first order model (r2 ≥ 0.99), indicating that parent and metabolites were equally bioavailable to undergo mineralization.

The percent recovered as 14CO2 equaled 10.4% by day 1 and increased to 60.8% by day 148.

Table 1: Fate of C10-13-LAS (Linear alkylbenzene sulfonate, average chain length 11.6) in aerobic sediment: Die-away study using Lytle Creek sediment (Study# 68316)

Time (days) Parent (Rf 0.36) Non-Polar Solids CO2 Total
Metabolite (Rf 0.57) Recovery
0.01 108.6±11.0 1.4 4±0.3 Not sampled 113.2±0.0
1 72.9±9.1 5.7 22.9±1.6 10.4±0.5 108.9±0.5
2 69.5±3.7 0 20.3±1.7 20.2±0.7 110±0.7
3 60.9±2.2 1.3±0.5 23.9±2.6 18.8±0.3 104.9±0.3
6 61.8±4.6 4 22.8±0.5 19.4±0.3 106±0.3
9 64.5±8.8 1.7 19.8±1.7 21.5±0.4 106.7±0.4
14 56.7±10.4 1.9 18.9±0.3 31.9±0.7 108.4±0.7
33 39.5±1.5 1±0.2 18.6±0.5 46.5±0.3 105.4±0.3
61 26.7±0.2 0.7±0.007 16.9±1.4 53.5±0.1 97.8±0.1
148 24.5±0.05 1.4±0.6 14.4±0.7 60.8±0.3 101.0±0.3
Abiotic (n=5) 107.5±3.7 Not detected 3±1.3 Not analyzed 110.6 ±2.1 
Validity criteria fulfilled:
yes
Conclusions:
C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) was aerobically biodegraded in sediment (Lytle Creek). After 148 days for biologically active samples, 60.8% was mineralized, 14.4% was associated with solids, 1.4% was metabolites, and 24.5% remained as parent. For abiotic samples mineralization was not analyzed, 3% was associated with solids and 107.5% remained as parent. The rate constants for primary biodegradation (compartment 1 and 2) and mineralization were found to be 1.5, 0.007 and 0.06 day-1 respectively.
Executive summary:

The biodegradation of C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) in sediment was evaluated in an aerobic die-away study conducted in a method similar to OECD guideline 308 and 314.

The inoculum (sediment) was collected from the Lytle Creek, Wilmington, Ohio. Radiolabeled test material (14C) was incubated with biotic and abiotic sediment samples under static conditions. The test material was added to the sediment to a final added concentration of 1.5 mg/kg dry weight and the test system was incubated for 148 days. The evolved 14CO2 was analyzed throughout the exposure duration. In addition, replicates of abiotic and biotic sediment were analyzed by parent, metabolites and non–extractable radioactivity.

The data collected from the abiotic samples was consistent and demonstrated that the parent remained stable throughout the test. In biotic samples, the parent LAS underwent primary degradation with the appearance of one non-polar metabolite (unidentified) with Rf 0.57. Test substance declined from 109% after 5 minutes to <27% by 61 days. Primary degradation was biphasic (two compartment first order model) with two pools of material exhibiting different degradation rates. The rates of primary degradation were 42.2 and 64.8% in Pool A and B, respectively. The rate constants for primary degradation were 1.5 and 0.007 day-1 and half-lives were 0.4 and 99 days in Pool A and B, respectively.

The percent 14CO2 was 10.4% by day 1 and increased to 60.8% by day 148 for biotic samples.14CO2 production was best described by a first order model, indicating that parent and metabolites were equally bioavailable to undergo mineralization.

Based on above results, C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) was aerobically biodegraded in sediment (Lytle Creek) at the end of 148 days exposure with about 60.8% being mineralized to CO2, 14.4% associated with solids, 1.4% as metabolites and 24.5% remained as parent.

This biodegradation in water sediment simulation study is classified as acceptable and is comparable to OECD guidelines 308 and 314.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jun.7, 2005 to Dec.16, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 314
Deviations:
no
GLP compliance:
no
Remarks:
The study was not originally conducted for REACH purposes.
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment
Details on source and properties of sediment:
Sediment was collected from the Ohio River near Cincinnati, Ohio at approximately river mile 476-477. The sediment was collected using a dredge and the top 2-3 cm layer of sediments was retained. The sediment was placed in plastic jars and kept on ice during transport and then stored in a 4°C cold room prior to the test. The sediment was characterized by the University of Wisconsin, Madison Soil & Plant Analysis Laboratory. The sediment consisted of 55% sand, 36% silt, and 9% clay. Organic content was 2.4%, total nitrogen content was 0.1% and pH was 7.6.
Duration of test (contact time):
92 d
Initial conc.:
1.5 other: mg/kg dry weight
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
other: Metabolites
Details on study design:
Overview of the experimental design

The radiolabeled test substance was incubated with biotic and abiotic sediment samples under static conditions. The sediment for the abiotic treatment was autoclaved for 90 min and amended with mercuric chloride at 1 g/L to inhibit microbial activity. The test systems consisted of replicate 1 mL samples of sediment with 0.1 mL overlying water in test tubes, which were individually dosed with the test substance at a final added concentration of 1.5mg/kg dry weight.

Four replicate samples for the biotic treatment were prepared per sampling interval and all the biotic samples were incubated together in a sealed dessicator, which contained a 50 mL beaker containing 20 mL of 1.5 N KOH to trap any evolved 14CO2 in the headspace. In addition, the dessicator was continuously purged with CO2-free air to maintain aerobic conditions and the effluent gas was passed through a gas trapping system consisting of one empty trap followed by three base traps containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap.

At each sampling, levels of evolved and dissolved 14CO2 were determined for the biotic treatments. In addition, replicates of abiotic and biotic sediment were analyzed for parent, metabolites and non-extractable radioactivity. (Refer figures 1 and 2 at page 10 of the study report (WTDS#68315) for diagrams of the test system and the analysis scheme).

TEST CONDITIONS
- Volume of test solution/treatment: 1 mL sediment with 0.1 mL overlying water
- Composition of medium: natural sediment, overlying water
- Additional substrate: no
- Solubilising agent: not used
- Test temperature: 22°C
- pH: not specified in the study report
- pH adjusted: no
- Aeration of dilution water: N/A
- Suspended solids concentration: N/A
- Continuous darkness: not specified in the study report
- Any indication of the test material adsorbing to the walls of the test apparatus: no

TEST SYSTEM
- Culturing apparatus: test tubes
- Number of culture flasks/concentration: 4 at each time point (biotic) and 2 at each point (abiotic)
- Method used to create aerobic conditions: CO2-free air continuously pumped through test system
- Measuring equipment: As per the details described in analytical methods.
- Test performed in closed vessels: yes (CO2-free air purged through system to measure evolved
14CO2 in traps)
- Details of trap for CO2: Details could be referred as per the overview of experimental design above.

SAMPLING
- Sampling frequency: Both (Biotic and abiotic) treatments were sampled after 15 min and after 1, 2, 3, 7, 10, 15, 36, and 92 days.

- Sampling method: At each sampling, the dessicator was opened to change the internal base trap and to recover sufficient samples for dissolved 14CO2, and for characterizing residual radioactivity.

- Sample storage before analysis: not specified in study report

STATISTICAL METHODS: The parent loss and mineralization data were fit to a variety of first order decay and production equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D software (version 4.01).
Compartment:
biologically active treatment at end of test
% Recovery:
75.8
St. dev.:
2
Compartment:
abiotic control measured at end of test
% Recovery:
105.8
St. dev.:
6.8
Key result
% Degr.:
42.1
St. dev.:
2.7
Parameter:
CO2 evolution
Sampling time:
92 d
Remarks on result:
other: Mean of biotic flasks; aerobic; normalized to 100% balance
Key result
% Degr.:
28.5
St. dev.:
3.7
Parameter:
other: associated with solids
Sampling time:
92 d
Remarks on result:
other: Mean of biotic flasks; aerobic; normalized to 100% balance
Key result
% Degr.:
0
Parameter:
other: non polar metabolites
Sampling time:
92 d
Remarks on result:
other: Mean of biotic flasks; aerobic; normalized to 100% balance
Key result
% Degr.:
29.8
St. dev.:
6.7
Parameter:
other: parent
Sampling time:
92 d
Remarks on result:
other: Mean of biotic flasks; aerobic; normalized to 100% balance
Key result
% Degr.:
99.5
St. dev.:
3.3
Parameter:
other: parent
Sampling time:
92 d
Remarks on result:
other: Mean of abiotic flasks; aerobic
Key result
% Degr.:
5.5
St. dev.:
1.9
Parameter:
other: associated with solids
Sampling time:
92 d
Remarks on result:
other: Mean of abiotic flasks; aerobic
Key result
Compartment:
sediment
DT50:
1.4 d
St. dev.:
0.2
Type:
other: two compartment first order model
Remarks on result:
other: Primary biodegradation; aerobic; compartment 1
Key result
Compartment:
sediment
DT50:
77 d
St. dev.:
0.001
Type:
other: two compartment first order model
Remarks on result:
other: Primary biodegradation; aerobic; compartment 2
Key result
Compartment:
sediment
DT50:
11.6 d
St. dev.:
1.9
Type:
other: first order
Remarks on result:
other: The rate of mineralization was determined using a first order model
Mineralization rate (in CO2):
0.06 d-1
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
Two metabolites were identified. The metabolite at Rf 0.70 reached a maximum of 4.7% by day 15 and subsequently disappeared. The metabolite at Rf 0.90 reached a maximum of 2.6% and disappeared by day 15.
Evaporation of parent compound:
no
Volatile metabolites:
not measured
Residues:
yes
Details on results:
Primary degradation: It was best described by a two compartment first order model (r2 ≥ 0.99). The process was biphasic with two pools (compartments) of material exhibiting different degradation rates. Pool A (compartment 1) presumably was readily bioavailable test material, in the aqueous phase. Pool B (compartment 2) presumably was less bioavailable test material, bound to solids (sorbed).

Primary degradation in pool A (compartment 1): 49.3±2.2%
Primary degradation in pool B (compartment 2): 49.9±2.2%

Mineralization (14CO2 production): It was best described by first order model (r2 ≥ 0.94) indicating that parent and metabolites were equally bioavailable to undergo mineralization.

The percent recovered as 14CO2 equaled 6% by day 1 and increased to 31.6% by day 92.

Table 1: Fate of C10-13-LAS (Linear alkylbenzene sulfonate, average chain length 11.6) in aerobic sediment: Die-away study using Ohio River sediment (Study#68315)

Time (days) Parent (Rf 0.45) Non-Polar Metabolite (Rf 0.70) Non-Polar Metabolite (Rf 0.90) Solids CO2 Total
Recovery
0.01 91.2±8.7 1.4±0.2 2.6±1.7 6.6±0.4 1.2±0.3 102.7±0.3
1 60.1±3.8 0.8±0.1 1.6±0.7 22.8±1.2 5.9±0.4 91.2±0.4
2 64.8±6.7 0.9±0.2 2.4±1.2 24.6±1.3 7.9±0.2 100.5±0.2
3 60.7±8.6 0.9±0.03 1.5±0.06 24.1±2.8 9.0±0.6 96.1±0.6
7 58±10.2 0.4±0.6 1.3±0.3 25.8±0.6 10±0.03 95.5±0.03
10 45.6±2.9 0 0.9±0.2 28.7±1.3 11.7±0.2 86.9±0.2
15 31.4±2.2 4.7±4.9 0 23.7±0.3 19.1±0.9 78.8±0.9
36 36.7±8.2 0 0 23.9±0.4 27.9±0.5 88.4±0.5
92 22.6±5.1 0 0 21.6±2.8 31.6±2.0 75.8±2.0
Abiotic 99.5±3.3 Not detected Not detected 5.5±1.9 Not analyzed 105.8±6.8
(n=10)

Biodegradation results in biotic flasks at 92 days were normalized to 100% mass balance as follows:

Mineralization: 31.6% to 42.1%

Solids: 21.5% to 28.5%

Metabolites: 0% to 0%

Parent: 22.6% to 29.8%

Mass Balance: 75.8% to 100%

Validity criteria fulfilled:
yes
Conclusions:
C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) was aerobically biodegraded in sediment (Ohio River). After 92 days for biologically active samples, 42.1% was mineralized, 28.5% was associated with solids, 0% was metabolites and 29.8% remained as parent. For abiotic samples mineralization was not analyzed, 99.5% remained as parent and 5.5% was associated with solids. The rate constants for primary biodegradation (compartment 1 and 2) and mineralization were found to be 0.5, 0.009 and 0.06 day-1 respectively.
Executive summary:

The biodegradation of C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) in sediment was evaluated in an aerobic die-away study conducted in a method similar to OECD guideline 308 and 314.

The inoculum (sediment) was collected from the Ohio River, Cincinnati, Ohio. Radiolabeled test material (14C) was incubated with biotic and abiotic sediment samples under static conditions. The test material was added to the sediment to a final added concentration of 1.5 mg/kg dry weight and the test system was incubated for 92 days. The evolved 14CO2 was analyzed throughout the exposure duration. In addition, replicates of abiotic and biotic sediment were analyzed by parent, metabolites and non-extractable radioactivity.

The data collected from the abiotic samples was consistent and demonstrated that the parent remained stable throughout the test. In biotic samples, the parent LAS underwent primary degradation with the appearance of two non-polar metabolites (unidentified) with Rf 0.70 and 0.90. Test substance declined from 91% after 15 minutes to <22% by 92 days. Primary degradation was biphasic (two compartment first order model) with two pools of material exhibiting different degradation rates. The two pools were the same size   but pool one was more readily bioavailable as indicated by the faster k1. The rates of primary degradation were 49.3 and 49.9% in Pool A and B, respectively. The rate constants for primary degradation were 0.5 and 0.009 day-1 and half-lives were 1.4 and 77 days in Pool A and B, respectively.

The percent 14CO2 was 6% by day 1 and increased to 31.6% by day 92 for biotic samples.14CO2 production was best described by a first order model, indicating that parent and metabolites were equally bioavailable to undergo mineralization. At the end of 92 days exposure, about 31.6% was mineralized to CO2, 21.6 % associated with solids and 22.6% remained as parent.

The mass balance in this study was found to be 75.8% which was normalized to 100%. Based on 100% mass balance normalization, 42.1% was mineralized to CO2, 28.5% was associated with solids, 0% was metabolites and 29.8% remained as parent.

Based on above results, C10-13 LAS (linear alkylbenzene sulfonate, average chain length 11.6) was aerobically biodegraded in sediment (Ohio River) at the end of 92 days exposure with about 42.1% being mineralized to CO2, 28.5 % associated with solids and 29.8% remained as parent.

This biodegradation in water sediment simulation study is classified as acceptable and is comparable to OECD guidelines 308 and 314.

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From June 10, 1992 to July 8, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Deviations:
no
GLP compliance:
yes
Remarks:
according to EPA Good Laboratory Practice Standards (40 CFR, Part 792)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The activated sludge was collected on June 10, 1992 from the Avondale Sewage Treatment Plant in Avondale, Pennsylvania, and from the Goose Creek Wastewater Treatment Plant in West Chester, Pennsylvania. Waste water was also collected twice a week immediately following the bar screen from Avondale plant and following primary clarification from Goose Creek plant.
- These sewage treatment plants receive predominantly domestic waste
- Storage conditions: Waste water was stored refrigerated in 50-gallon drums. The drums were equipped with mechanical mixers to keep suspended solids uniformly distributed. The collected sludge was aerated until use
- Storage length: Maximum storage time of a single batch of wastewater was 5 d. The sludge was used as soon as possible after the collection.
- Preparation of inoculum for exposure: The sludge was screened to remove large clumps and aerated. TSS levels were determined and based on these readings, each sludge was distributed to two CAS units at a concentration of approx. 2,500 mg TSS/L. The waste water was filtered through a 20 mesh stainless steel screen and refrigerated in 50-gallon drums.
- Concentration of sludge: Not reported
- Initial cell/biomass concentration: Not reported
Duration of test (contact time):
23 d
Initial conc.:
1.5 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 6 L
- Composition of medium: Each aeration basin contained
6 L activated sludge
Feed solution (consisting of approx. 910 mL of wastewater and approx. 90 mL feed solution pumped at flow rate of 1000 mL/h)
- Test temperature: 21.0-22.5°C
- pH: 7.0-7.8
- pH adjusted: No
- COD (mg/L): 162-281 (influent); 45-51 (effluent)
- BOD5 (mg/L): 67-140 (influent); 5-11 (effluent)
- Aeration: Yes, aeration basins were aerated by gas dispersion tubes using compressed air
- Suspended solids concentration in test CAS units (TSS): 99-197 mg/L (influent); 2478-3369 mg/L (mixed liquor); 14-20 mg/L (Effluent)
- Any indication of the test material adsorbing to the walls of the test apparatus: No

TEST SYSTEM
- CAS UNITS: Each CAS unit consisted of
Mixing chamber: Served to mix the incoming wastewater and test feed solution before it entered the aeration basin
Aeration basin: Contained 6L of the appropriate activated sludge and was aerated by two gas dispersion tubes
Cylindrical clarifier: The aeration basin discharged to a 2L cylindrical clarifier, which was stirred by a shaft mixer at approx. 2 rpm. The clarifier had a recycle discharge in the bottom and a side tube for effluent discharge.
- The volume of sludge wasted in any unit did not exceed 1 L per day
- Flow rate of wastewater into mixing chamber: 15.2 mL/min via peristaltic pump
- Flow rate of test material into mixing chamber: 1.5 mL/min via peristaltic pump.
- Influent flow rate to the aeration basin: 1000 mL/h (consisting of approx. 910 mL of wastewater and approx. 90 mL feed solution) via an overflow tube
- Sludge recycling period: The recycle period consisted of a continuous 15 min on and 15 min off. A 15-min interval recycle timer was used to control the recycle pump
- Sludge blanket height: 1.85-2.85 inch
- Hydraulic residence time (HRT): 6.0±0.5 h
- Sludge retention time (SRT): 5-12 d (units maintained with Avondale wastewater); 8-19 d (units maintained with Goose Creek wastewater)
- Number of CAS units: Four CAS units were used; pairs of treated and untreated (controls) received sewage wastewater and sludge from Avondale Plant and from Goose Creek Plant, respectively.
- Test performed in closed vessels: no

OPERATION OF CAS UNITS: Operation of the CAS units was divided into three phases:
- Stabilization: Following initial set-up, the CAS units were allowed to stabilize for 21 d to allow the mixed liquor to adjust to laboratory condition. During this Stabilization period, all units received wastewater and deionized water without test substance present.
- Acclimation: The stabilization period was followed by an acclimation period lasting for 11 d. The purpose of acclimation was to pre-expose the microbial population to the test substance. During this time, the units received wastewater mixed with a prepared test feed solution at the specified concentration of 1.5 mg active/L with a radiochemical concentration of 1. 8 µCi/L or deionized water in the control units
- Removal: The preparation of the individual test feed solutions followed the procedure used during acclimation. The nominal influent concentration remained same. During the removal period, influent, aeration mixed liquor and effluent samples were removed five times weekly and tested for the level of radioactivity present

SAMPLING
- Sampling frequency: Samples were collected on six days and 5 times/week from the test units during acclimation and removal period respectively
- Sampling method during acclimation period: Triplicate 1 mL aliquots of the influent sample, triplicate 5 mL aliquots from the effluent sample and triplicate 2 mL aliquots from the aeration basin were collected
- Sampling method during removal period: Triplicate 1 mL aliquots of the influent sample, triplicate 1 mL aliquots from the aeration basin, triplicate 2 mL aliquots from the aeration basin and triplicate 5 mL aliquots from the effluent samples were collected.
The treatment of samples and further analysis is provided under section ‘Details in analytical method’ above.
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, received wastewater and deionized water at the same target flow rates
- Abiotic sterile control: No
- Toxicity control: No

STATISTICAL METHODS: Not reported
Reference substance:
not required
Test performance:
The addition of the test substance at a concentration of 1.5 mg active/L did not have an adverse effect on the overall CAS system performance.
% Degr.:
88.1
St. dev.:
1.8
Parameter:
radiochem. meas.
Sampling time:
12 d
Remarks on result:
other: Overall removal during 12 day removal period of the CAS unit (Inoculum source: Avondale sewage treatment) plant
% Degr.:
88.4
St. dev.:
1.8
Parameter:
radiochem. meas.
Sampling time:
12 d
Remarks on result:
other: Over all removal during 12 day removal period of the CAS unit (Inoculum source: Goose Creek treatment plant)
Transformation products:
not measured
Details on transformation products:
Not examined
Evaporation of parent compound:
no
Volatile metabolites:
not measured
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: None

TEST MATERIAL REMOVAL
- During acclimation phase
Inoculum source - Avondale sewage treatment: 90.8% + 1.7 (acidified effluent)
Inoculum source - Goose Creek treatment plant: 91.8% + 1.5 (acidified effluent)
- During removal phase
Inoculum source - Avondale sewage treatment: 88.1 + 1.8% (filter filtrate effluent)
Inoculum source - Goose Creek treatment plant: 88.4 + 1.8% (filter filtrate effluent)

EFFLUENT PARTITIONING BEHAVIOR OF THE TEST MATERIAL
Inoculum source - Avondale sewage treatment: 43.9% (mean) 14C present in the solid phase; 56.1% (mean) 14C present in the liquid phase
Inoculum source - Goose Creek treatment plant: 58.8% (mean) 14C present in the solid phase; 41.2% (mean) 14C present in the liquid phase.
For details, refer to ‘table 1’and ‘table 2’ under ‘any other information on results’.

RESULTS OF SUPPLEMENTARY EXPERIMENT (% efficiency):
Inoculum source - Avondale sewage treatment: 81.6, 85.2, 81.0, 71.2 and 59% at sludge levels of 0.5,1, 2, 3, 4 mL respectively.
Inoculum source - Goose Creek treatment plant: 80.4, 85.3, 80, 79.2 and 70.8 % at sludge levels of 0.5,1, 2, 3, 4 mL respectively.
Further details are provided in the study report.

Table 1: Continuous Activated Sludge (CAS) Test of Monoethanolamine: Effluent removal and partitioning data (study # 35393) (inoculum source: Avondale sewage treatment plant)

Study #35393 (inoculum source: Avondale sewage treatment plant)

Time (hours)

Mixed Liquor balance*

Effluent partitioning**

 

Effluent removal (during removal phase)

Solids (%)

Liquid (%)

Solids (%)

Liquid (%)

Unacidified effluent % removal

Acidified Effluent %removal

Filter/filtrate effluent % removal

24

51.9

1.4

31.8

68.2

82.1

87.7

89.1

48

51.7

1.2

43.1

56.9

76.4

85.1

86.6

72

60.3

2.2

30.5

69.5

79.9

84.4

85.8

96

58.2

1.3

47.2

52.8

77.5

87.5

86.8

120

67.3

1.2

50.1

49.9

76.6

84.1

86.5

144

-

-

-

-

84.4

89.7

-

168

-

-

-

-

83.5

89.7

-

192

50.6

1.4

45.3

54.7

84.8

88.6

88.7

216

76.1

1.5

49.3

50.7

84.2

89.8

89.8

240

62.1

1.7

35.7

64.3

82.6

87.2

87.9

264

44

1.9

51

49

83.4

88.5

88.5

288

56.8

0.6

55.3

44.7

83.1

91.3

91.6

Mean

57.9

1.4

43.9

56.1

81.5±3.1

87.8±2.2

88.1±1.8

Monoethanolamine (CAS # 141-43-5).

 

Table 2: Continuous Activated Sludge (CAS) Test of Monoethanolamine: Effluent removal and partitioning data (study # 35393) (inoculum source: Goose Creek treatment plant)

Study #35393 (inoculum source: Goose Creek treatment plant)

Time (hours)

Mixed Liquor balance

Effluent partitioning*

Effluent removal (during removal phase)

Solids (%)

Liquid (%)

Solids (%)

Liquid (%)

Unacidified effluent % removal

Acidified Effluent %removal

Filter/filtrate effluent % removal

24

56.2

0.7

49.1

50.9

85.2

89.1

89.8

48

55.9

0.5

55

45

84.7

88.1

89.1

72

59.9

0.9

35.5

64.5

85.7

88.7

89.4

96

61.7

0.4

58

42

86.1

90.9

91.1

120

62.4

0.5

64

36

85.3

88.5

89.4

144

-

-

-

-

83.8

88.1

-

168

-

-

-

-

80.2

84.5

-

192

65.7

0.8

59.3

40.7

84.4

87.2

88.4

216

76

0.6

67.6

32.4

81.2

85.2

85.6

240

95.3

0.7

64.3

35.7

83.4

85.1

85.7

264

45

0.9

64.9

35.1

85.3

87.9

88

288

76.7

0.5

70.1

29.9

83.5

87

87.3

Mean

65.5

0.6

58.8

41.2

84±1.8

87.5±1.8

88.4±1.8

Monoethanolamine (CAS # 141-43-5).

*The mixed liquor balance determines the % of 14C present in the acidified liquid or solid phase relative to the total unacidified, efficiency corrected mixed liquor counts.

**Effluent partitioning is based relative to counts after acidification

Table 3: Mean percent removal of TSS, COD and BOD5 (calculated by comparison of the TSS and COD removals for the control units with the respective test substance units) (Study #35393)

Mean percent removal (Study #35393)

TSS

COD

BOD5

Control Unit (Test system source: Avondale sewage treatment plant

Testing phase

85.9

76.3

93.2

Total study

89.3

80

93.2

Test Unit (Test system source: Avondale sewage treatment plant

Testing phase

82.1

73.4

91.9

Total study

87.9

79.1

92.2

Control Unit (Test system source: Goose Creek treatment plant

Testing phase

72.4

56.9

86.1

Total study

78.1

65

87.9

Test Unit (Test system source: Goose Creek treatment plant

Testing phase

76.4

59.1

90.6

Total study

83

69.3

91.9

 

Validity criteria fulfilled:
yes
Conclusions:
The removal of Monoethanolamine, tested at 1.5 active mg/L, was 88% in a Continuous Activated Sludge (CAS) study. This result is the mean of 2 CAS units that used activated sludge from 2 different sewage treatment plants (Avondale, 88.1%; Goose Creek, 88.4%).
Executive summary:

A Continuous Activated Sludge (CAS) study was conducted on Monoethanolamine, following the OECD Guideline 303 A (Simulation Test - Activated Sludge Units). The test concentration was1.5 mg/L. The test material was a combination of radiolabeled and unlabeled monoethanolamine. Two sources of inoculum (activated sludge) were used:  from the Avondale Sewage Treatment Plant in Avondale, Pennsylvania, and from the Goose Creek Wastewater Treatment Plant in West Chester, Pennsylvania.  Waste water was also collected from both sewage treatment plants.

Four CAS units were used;a control and test unit received sewage wastewater and sludge either from the Avondale Plant, or the Goose Creek Plant, respectively. Each CAS unit had a mixing chamber, aeration basin and cylindrical clarifier. Following stabilization (21 days) and acclimation period (11 days), the removal phase (12 days) was conducted. Samples (influent, aeration mixed liquor and effluent) were collected five times weekly during the removal phase and tested for the level of radioactivity present.

The mean removal of Monoethanolamine was 88% in the Continuous Activated Sludge (CAS) study.  Removal was 88.1% (with Avondale sludge) and 88.4% (with Goose Creek sludge).

No adverse effects on the overall CAS system performance were observed during any phases of the study period.

This biodegradation simulation test satisfied the guideline requirements for the OECD303 Aguideline.

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 26, 1989 to November 18, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD 314B guideline (biodegradation in activated sludge test)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA Good Laboratory Practice Standards (40 CFR, Part 792)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
Not applicable
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from the Avondale Sewage Treatment Plant, Avondale, Pennsylvania. This STP receives predominantly domestic waste.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: Not reported
- Preparation of inoculum for exposure: Not reported
- Pretreatment: Screened through a 2 mm sieve. The sludge was adjusted to this concentration by settling and decanting the appropriate quantity of supernatant.
- Concentration of sludge: 2500 mg/L of TSS
- Initial cell/biomass concentration: Not reported
- Water filtered: Not applicable
- Type and size of filter used, if any: Not applicable
Duration of test (contact time):
17 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 1 L
- Composition of medium: Each 1 L test flask contained:
1 L activated sludge (2500 mg SS/L)
Test chemical stock solution (1 mg/L)
- Additional substrate: None
- Solubilising agent: Not used
- Test temperature: 22.0-23.6 ºC
- pH: Not reported
- pH adjusted: Not reported
- Aeration of dilution water: Yes, please see below
- Suspended solids concentration: Approx. 2500 mg/L
- Continuous darkness: Not reported
- Any indication of the test material adsorbing to the walls of the test apparatus: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L Erlenmeyer glass flasks
- Number of culture flasks/concentration: Three
- Method used to create aerobic conditions: The test solutions were placed on rotary platform shaker were aerated with by connecting to CO2-free air source. The aeration tube in each flask was extended below the sludge level. The speed of the shaker was adjusted to keep the sludge solids in suspension. Moreover, air was purged through the scrubbing solutions from a pressurized air source at a flow rate of approx. 55 mL/min, equivalent to 1-2 bubbles/second.
- Measuring equipment: Carbon dioxide measuring apparatus consisted of CO2 scrubbing apparatus (Two empty 1 L plastic bottles, one each to prevent backflow and overflow into the air line and test containers; Five 1 L plastic bottles containing 700 mL 10 N NaOH) connected in series with Tygon tubing to a pressurized air source and a carboy.
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used:
(a) Trap for CO2: First alkali trap in the 14CO2 train is removed and sub-sampled immediately or stored tightly capped. The two remaining traps are moved one slot closer to the flask and a new trap is added to the third slot. 1 mL samples are withdrawn from the first external base trap and counted in 20 mL of Cab-O-Sil.
(b) Trap for volatile organics: Potential volatilisation was evaluated by placing vapor traps in the purging line between the test flask and the alkali trap. The vapor trap consisted of a sorbent tube containing a residue free XAD resin. Each sorbent tube was divided into two compartments by glass wool. At each volatilization sampling period each sorbent tube was removed and replaced with a freshly prepared tube.

SAMPLING
- Sampling frequency: At 1, 2, 4, 8 and 24 h of Day 1 and on Day 2, 3, 5, 7, 14 and 17
- Sampling method:
(a) For CO2 determination: The first 14CO2 alkali trap in the individual trains was removed and a 1mL aliquot was counted by liquid scintillation counting (LSC) in 20 mL of Cab-O-Sil. The remaining traps were moved one slot closer to the test flask and a new trap was added to the third slot. At the same time base traps are sampled, 10 mL sludge samples are withdrawn by syringe and filtered through 0-0.45 µm filters. The filter sets were washed with a total of 5 mL IPA/water (50/50) mixture, air dried, and counted in 20 mL of 3A cocktail to quantitate radioactivity in the microbial biomass.
(b) For volatilization sampling: The sorbent tube’s containing resins were periodically sampled for 14C analysis (by transferring into LSC vials, cocktailed in 3A cocktail) and replaced with freshly prepared tubes. The scheduled 4 h vapor trap sampling occurred at 4.75 h.
(c) To determine soluble/sorbed radioactivity, a 20 mL sample of sludge was withdrawn from each flask. TSS was determined using 5 mL aliquots and remaining sludge was centrifuged and 1 mL supernatant aliquots were cocktailed in 3A cocktail and analysed for 14C.
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not reported
- Other: The study was terminated by adding one 1 mL concentrated HCl to the flasks and one final set of samples was collected and treated as above.
CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes, the test system consisted of 1L of autoclaved deionized water in a 2 L flask. (14C Substrate = 87µL, unlabelled sustrate = 1 mL)
- Toxicity control: No

STATISTICAL METHODS: Percent 14CO2 production vs time was analysed by the following empirical model:

14CO2 = a (1 - e(-k(t-c)))
where,
a = Extent of 14CO2 production (%)
k1 = First order rate constant (day-1)
t = Time of incubation (days)c = Lag period, if any (days). The constants a and k1 along with 95 % confidence intervals were generated for each treatment
Reference substance:
not specified
Compartment:
other: water, material (mass) balance
% Recovery:
92.3
St. dev.:
8
% Degr.:
83.1
St. dev.:
6.6
Parameter:
CO2 evolution
Sampling time:
17 d
Remarks on result:
other: mean of flasks 1, 2, 3
% Degr.:
77.1
Parameter:
CO2 evolution
Sampling time:
17 d
Remarks on result:
other: In flask 1
% Degr.:
90.1
Parameter:
CO2 evolution
Sampling time:
17 d
Remarks on result:
other: In flask 2
% Degr.:
82
Parameter:
CO2 evolution
Sampling time:
17 d
Remarks on result:
other: In flask 3
Other kinetic parameters:
first order rate constant
Transformation products:
not measured
Details on transformation products:
Transformation products were not determined in the study
Evaporation of parent compound:
no
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS: Not determined in the study.

MINOR TRANSFORMATION PRODUCTS: Not determined in the study.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: Not reported

EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not applicable
- % of applied amount at end of study period: Not applicable

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not applicable
- % of applied amount at end of study period: Not applicable

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 77.1, 90.1 and 82.0% in flask 1, 2 and 3 respectively.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 0 in all the test flasks.

STERILE TREATMENTS: not used

RESULTS OF SUPPLEMENTARY EXPERIMENT: None

Table 1 Biodegradation and Volatilization Test of Ethanol amine (study # 35292): FLASK #1

Time Solution % 14C Biomass % 14C Total % 14CO2 per sampling * Cumulative Resin Trap % 14C Mass Balance
1 hr 2.8 16.1 21.5 0 40.4
2 hr 7.1 20 18.5 0 45.6
4 hr 5.9 15.9 25.6 0 47.4
8 hr 9.1 13.5 33.7 0 56.3
1 day 4.3 14.3 43.6 0 62.2
2 days 2.2 12 52.3 0 66.5
3 days 2.4 12.4 58.5 0 73.3
5 days 2.4 9.6 67.9 0 79.9
7 days 2.4 6.8 76.1 0 85.3
14 days 2.7 11.9 71.2 0 85.8
17 days 3.6 4.6 77.1 0 85.3

Table 2 Biodegradation and Volatilization Test of Ethanol amine (study # 35292): FLASK # 2

Time Solution % 14C Biomass % 14C Total % 14CO2 per sampling * Cumulative Resin Trap % 14C Mass Balance
1 hr 2.2 18.4 20.3 0 40.9
2 hr 6.6 21.8 19.1 0 47.5
4 hr 3.9 18.9 22 0 44.8
8 hr 10.9 18.6 30.4 0 59.9
1 day 4.3 16.1 42.7 0 63.1
2 days 2 13.9 51.5 0 67.4
3 days 2.5 13.1 58.1 0 73.7
5 days 3.7 11.9 71.6 0 87.2
7 days 2.3 8.4 78.1 0 88.8
14 days 3.2 6.5 88.9 0 98.6
17 days 4.1 7 90.1 0 101.2

Table 3 Biodegradation and Volatilization Test of Ethanol amine (study # 35292): FLASK # 3

Time Solution % 14C Biomass % 14C Total % 14CO2 per sampling * Cumulative Resin Trap % 14C Mass Balance
1 hr 1.8 14.4 21.6 0 37.8
2 hr 9.1 21.1 21.5 0 51.7
4 hr 2.6 19.5 25.9 0 48
8 hr 2.8 15.9 32 0 50.7
1 day 3.9 15.7 38.6 0 58.2
2 days 1.8 13.1 47.8 0 62.7
3 days 4.8 11.9 54.7 0 71.4
5 days 4.6 9.8 65.2 0 79.6
7 days 4.2 8.5 71.7 0 84.4
14 days 2.7 7.5 82 0 92.2
17 days 2.5 6.1 82 0 90.6

*The sum of cumulative trap % 14CO2, and volatile base % 14CO2.

Validity criteria fulfilled:
not applicable
Conclusions:
Ethanolamine underwent mineralization in an activated sludge simulation test. At Day 17, the %CO2 produced was 83.1% (mean of 3 flasks). The rate constant for mineralization in activated sludge was 0.04 day-1 (mean of 3 flasks).
Executive summary:

A simulation of the mineralization of ethanolamine in activated sludge was conducted in accordance with the OECD 314B guideline.  Radiolabeled ethanolamine (14C) was tested at 1 mg/L. The inoculum was activated sludge (2500 mg/L TSS) obtained from a municipal sewage treatment plant that receives predominantly domestic waste (the Avondale STP). The test treatments were measured in triplicate.

The cumulative percent of theoretical 14CO2 produced was 83.1% at Day 17 (mean of three flasks) 

The rate constant for mineralization of ethanolamine in activated sludge was 0.04 day-1 (average of the 3 flasks, ranging from 0.03-0.07 day-1).

 

This mineralization simulation test satisfied the guideline requirements for the OECD 314 B simulation tests to assess the biodegradability of chemicals discharged in wastewater.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 25, 1992 to Aug. 20, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 314D. Deviations, reliability, and validity evaluated against current OECD 314D (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
according to EPA principles of GLP
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on source and properties of surface water:
- Details on collection: River water was collected from the East Branch of the Brandywine Creek in Downingtown, Pennsylvania (just below the surface) on June 23, 1992 using plastic containers.
- Storage conditions: Water samples were composited and stored under refrigeration.
- Storage length: 2 d
- Temperature (°C) at time of collection: Not reported
- pH at time of collection: 8.0
- Total hardness: 24.4 mg/L
- Total organic carbon: 15 mg/L
- Organic nitrogen: 0.49 mg/L
- Ammonia as N: <0.10 mg/L
- Nitrite as N: <0.10 mg/L
- Nitrate as N: 3.3 mg/L
- Biomass: 5.6 x 10(3) CFU/mL
- TSS: 1 mg/L
Duration of test (contact time):
50 d
Initial conc.:
300 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 1 L of river water
- Composition of medium: Each 2 L test flask contained:
1 L river water
Appropriate volume of stock solution to obtain 300 µg active/L
- Test temperature: 21.9 - 23.0°C
- pH: Not reported
- Aeration of dilution water: Continuous aeration with CO2-free air.
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L Erlenmeyer flasks
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: The flasks were placed on a rotary platform shaker (100 to 150 rpm) and aerated continuously with a CO2 free air source. Air was sparged through the scrubbing train at a constant rate adequate to provide 1-2 bubbles/sec in the alkali traps. The CO2 scrubbing apparatus consisted of: (a) One empty 1 L plastic bottle, to prevent backflow; (b) Five 1 L plastic bottles containing 700 mL 10 N NaOH and (c) One empty 1 L plastic bottle to prevent overflow of alkali into the test containers connected in series with Tygon tubing to a pressurized air source (approx. 10-15 psi).
- Test performed in closed vessels: Yes
- Details of trap for CO2 and volatile organics if used: Glass bottles approx. 4 oz size containing 100 mL of 1.5 N KOH.
- Other: The study was terminated on Day 50 by adding 1 mL concentrated HCl to the flasks and shaking was continued for 3 d. After this final incubation, duplicate 10 mL water samples from all flasks were assayed by LSC in 10 mL Triton X Cocktail. Duplicate 1 mL samples from all three alkali traps were taken and counted by LSC in 20 mL Cab-O-Sil Cocktail.

SAMPLING
- Sampling frequency: On Day 1, 3, 5, 7, 10, 14, 21, 28, 42 and 50
- Sampling method: 1 mL samples were collected from the first alkali trap and counted by LSC in 20 mL Cab-O-sil. At the same time, base traps are sampled; 10 mL water samples were withdrawn and filtered through 0.2 µm filters. The filter sets were washed with 5 mL IPA/water (50/50) mixture, air dried and counted by LSC in 20 mL 3A Cocktail to quantitate radioactivity in the microbial biomass.
The filtrate was treated as described in "Details on analytical methods" above.
- Sample storage before analysis: Not reported

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: No
- Toxicity control: No

STATISTICAL METHODS
Percent 14CO2 production vs time was analyzed by the following empirical model:
14CO2 = a (1 - e(-k(t-c)))
where,
a = Extent of 14CO2 production (%)
k = First order rate constant (day-1)
t = Time of incubation (days)
c = Lag period, if any (days).
The constants a and k along with 95% confidence intervals were generated for each treatment and control. A graph illustrating 14CO2 production vs time was plotted for each test flask.
Reference substance:
other: d-glucose at a concentration of 300 µg/L (0.45 µCi/L).
Compartment:
other: water, material (mass) balance
% Recovery:
94.5
St. dev.:
6.4
% Degr.:
96.5
Parameter:
CO2 evolution
Sampling time:
50 d
Remarks on result:
other: Flask 1
% Degr.:
82.4
Parameter:
CO2 evolution
Sampling time:
50 d
Remarks on result:
other: Flask 2
% Degr.:
81.8
Parameter:
CO2 evolution
Sampling time:
50 d
Remarks on result:
other: Flask 3
% Degr.:
86.9
St. dev.:
8.3
Parameter:
CO2 evolution
Sampling time:
50 d
Remarks on result:
other: Mean of Flask 1, 2 and 3
Compartment:
water
DT50:
1.06 d
St. dev.:
0.09
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Other kinetic parameters:
first order rate constant
Transformation products:
not measured
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS: Not determined in the study.

MINOR TRANSFORMATION PRODUCTS: Not determined in the study.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 96.5, 82.4 and 81.8 in Flask 1, 2 and 3 respectively.
For details see ‘Table1’ below in the ‘Any other information on results incl. tables’ section.
Results with reference substance:
MINERALISATION
- % of applied radioactivity present as CO2 at end of study (day 50): 78.4% (Mass balance on Day 50: 87.8%)
(mineralization on Day 42 was 90.9%, with a mass balance of 95.1%.)

MASS BALANCE
- Final mass balance: 97.3%
For details see ‘Table 2’ below in the ‘Any other information on results incl. tables’ section.

Table 1: Biodegradation Test of Monoethanolamine (14C-MEA)

Flask #1 (study # 35326)

Day

%T14CO2

Biomass %14C

Solution %14C

Mass Balance

1

3.9

3

101.1

108.0

3

63.1

15.7

15.9

94.7

5

73.2

7.9

12.5

93.6

7

77.6

8

9.6

95.2

10

80.3

5.4

10.7

96.4

14

80.5

4.3

8.9

93.7

21

76.3

4.3

7.4

88.0

28

81.1

3.4

8.4

92.9

42

92.2

2.9

4.8

99.9

50*

96.5

2.7

2.6

101.8

Flask #2 (study # 35326)

Day

%T14CO2

Biomass %14C

Solution %14C

Mass Balance

1

4.4

4.1

83.8

92.3

3

61.5

17.7

14.4

93.6

5

67.1

8.3

14.1

89.5

7

68.1

7.7

12.4

88.2

10

77.5

6.9

10.1

94.5

14

72.5

5.6

9.1

87.2

21

70.2

5.1

8

83.3

28

79.6

3.7

6.9

90.2

42

81.3

3.4

4.8

89.5

50*

82.4

3.6

6.1

92.1

Flask #3 (study # 35326)

Day

%T14CO2

Biomass %14C

Solution %14C

Mass Balance

1

4.3

3.6

102.6

110.5

3

56

19.8

17.3

93.1

5

66.4

9.2

15.4

91.0

7

69.5

7.8

13.8

91.1

10

69.6

6.7

10.1

86.4

14

76.9

5.6

8.7

91.2

21

80.5

4.3

8.4

93.2

28

79.2

3.5

7.6

90.3

42

87.3

3.6

5.3

96.2

50*

81.8

2.1

5.8

89.7

 

Table 2: Biodegradation Test of d-Glucose (14C D-glucose) (study # 35326)

Day

%T14CO2

Biomass %14C

Solution %14C

Mass Balance

1

43.6

37.3

14.5

95.4

3

70.8

13.2

12.5

96.5

5

69.1

8.8

10.8

88.7

7

78.2

5.7

9.9

93.8

10

83.1

5.5

7.4

96.0

14

86.9

5

6.3

98.2

21

81.8

3.9

4.8

90.5

28

83.8

3.5

5.6

92.9

42

90.9

2.1

2.1

95.1

50*

78.4

2.1

7.3

87.8

*Flask was acidified following completion of sampling.

Test material was MEA (Monoethanolamine), CAS # 141-43-5.

The mass balance was calculated by adding the %14CO2, Biomass%14C and solution %14C.

Validity criteria fulfilled:
yes
Conclusions:
Monoethanolamine (MEA) underwent complete mineralization in surface water. At Day 50, the %CO2 produced was 86.9% (mean of flask 1, 2 and 3). The rate constant for mineralization in surface water was 0.59 day-1 (mean of flask 1, 2 and 3).
Executive summary:

A simulation of the mineralization of Monoethanolamine (MEA) in surface water was conducted under aerobic conditions in accordance with the OECD 314D guideline. Monoethanolamine was tested at 300 µg/L. Radiolabeled monoethanolamine was added at 0.45 µCi/L.

The river water was collected from Brandywine Creek in Downingtown, Pennsylvania.The test treatments were measured in triplicate. Radiolabeled d-Glucose(14C) served as a reference control.

After 50 d, the cumulative percent of theoretical 14CO2 produced was 86.9% (mean of flasks 1, 2 and 3). The mass balance was 94.5%. Normalized to 100% mass balance, mineralization of monoethanolamine at day 50 was 91.8%.

The rate constant for mineralization of Monoethanolamine (MEA) in surface water was 0.59 day-1(mean of flasks 1, 2 and 3). This corresponds to a half-life of 1.06 days.

This biodegradation simulation test satisfied the guideline requirements for the OECD 314 D simulation tests to assess the biodegradability of chemicals discharged in wastewater.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Sept. 20, 1996 to July 27, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Deviations:
no
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: Domestic raw wastewater from Polk Run WWTP in Loveland, OH. Activated sludge plant received approximately 5% industrial wastewater and 95% domestic water.
Details on inoculum:
- Source of activated sludge: For each CAS unit to be operated, ~3.0 L of activated sludge mixed liquor was collected from Polk Run, Loveland, OH WWTP (Cincinnati MSD). Polk Run is a 6 million gallon per day (MGD) activated sludge plant that received approximately 5% industrial wastewater (95% domestic). The sludge was collected at a point adjacent to the effluent weir of one of the four aeration basins at Polk Run and was returned to the lab and placed in the laboratory CAS aeration unit within 90 minutes. The additional CAS system volume was filled with approximately 3-L of tap water.
- Storage conditions: Each CAS system (#1-Control CAS & #2-Test CAS) was fed raw wastewater that was collected from the Polk Run POTW and stored at ESD in a mechanically-mixed 200 L carbouy in an adjacent constant temperature (CT) box maintained at 4-10°C.
- Storage length: Fresh wastewater was collected 2 times per week from Polk Run in order to maintain appropriate biological and organic loading associated with domestic WWTP's.
Duration of test (contact time):
2 mo
Initial conc.:
1 mg/L
Based on:
test mat.
Initial conc.:
3 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
other: Removal by Sorption to MLSS
Details on study design:
The focus of the study was to determine if effluent concentration was dependent or independent of influent concentration during secondary activated sludge wastewater treatment for linear alkylbenzenesulfonate (LAS). Two CAS systems (control unit and test unit) were operated at a Domestic raw wastewater from Polk Run WWTP in Loveland, OH was used as a feed source for both CAS units. The control unit received only the domestic wastewater as a feed source. The testing unit was fed domestic wastewater along with 1, 3 and 10 mg/L concentrations of LAS (non-labeled LAS with a tracer of 14C radiolabeled LAS). Only one concentration was tested at a time with an equilibration period of approximately one month between testing concentrations. Initially a 2-phenyl isomer of 14C-C12LAS was tested at 1 mg/L mixed with a small amount of non-radiolabeled commercial mixture of LAS. All further testing was accomplished using a 3,4,5-phenyl isomer of 14C-C12LAS mixed with appropriate amounts of the non-labeled commercial LAS mixture. Mass balances were conducted for each system for adsorption to MLSS, mineralization, and overall system removal using liquid scintillation counting (LSC). Radio-thin layer chromatography (Rad-TLC) and combustion of mixed liquor suspended solids (MLSS) was used to determine the extent of parent and metabolite loss from the system, adsorbed onto, and incorporated into MLSS.

TEST CONDITIONS
- Pump: Peristaltically through platinum-lined silicone tubing
- Nominal flow rate for waste water feed solution: 8.3 mL/min
- Hydraulic retention time (HRT): 6 hours
- Solids retention time (SRT): 10-14 days
- Volume of test solution/treatment: 3.0 L of activated sludge mixed liquor and additional 3.0 L of tap water
- Equilibration time: Two and one half weeks
- Procedure: The CAS aeration basin was mixed and aerated with unfiltered laboratory air. Every half hour settled sludge was returned to the aeration basin via an airlift system. All off-gasses were trapped in two sealed gas traps in series, containing monoethanolamine. The primary and secondary trap volumes were 500 mL and 300 mL, respectively. The wastewater feed solution was pumped into CAS units (control unit# 1 and test unit# 2) and allowed to equilibrate for two and one half weeks. The n, 14C-C12LAS was pumped at a nominal flow rate of ~0.4 mL/minute (5% of total flow) and the non-labelled test substance was pumped at nominal rate of 8.0 mL/min to Test CAS unit# 2. Stabilization of the radiolabeled material was determined by liquid scintillation counting (LSC) analyses of the various compartments in the CAS systems. Once equilibration was achieved, a five-day testing period was begun for that test level.

SAMPLING
- Sampling frequency: Daily
- Sample storage before analysis: Duplicate samples from the MLSS (centrifuged sample pellet, minus supernatant- study days 1, 3, & 5), radiolabeled dosing stock solutions, and CAS final effluents were frozen at ~80°C.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION CONTROL AND BLANK SYSTEM
- Inoculum blank: CAS unit# 1 was fed with only domestic raw wastewater from Polk Run WWTP in Loveland, OH. It was considered as control unit.
- Abiotic sterile control: No
- Toxicity control: No
Compartment:
entire system
% Recovery:
100
Key result
% Degr.:
99.44
St. dev.:
0.2
Parameter:
radiochem. meas.
Sampling time:
5 d
Remarks on result:
other: % parent removal at 1 mg/L
Key result
% Degr.:
99.36
St. dev.:
0.2
Parameter:
radiochem. meas.
Sampling time:
5 d
Remarks on result:
other: % parent removal at 3 mg/L
Key result
% Degr.:
99.37
St. dev.:
0.2
Parameter:
radiochem. meas.
Sampling time:
5 d
Remarks on result:
other: % parent removal at 10 mg/L
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
No.:
#6
No.:
#7
No.:
#8
Details on transformation products:
8 polar metabolites and 2 non polar metabolites were identified in the study. The percentage of each metabolite is provided in "Any other information on results incl. tables" section.
Evaporation of parent compound:
no
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: No

MAJOR TRANSFORMATION PRODUCTS: 8 polar metabolites and 2 non polar metabolites were identified in the study. The percentage of each metabolite is provided in "Any other information on results incl. tables" section.

PARENT REMOVAL
- 99.44, 99.36 and 99.37% at nominal concentrations of 1, 3, and 10 mg/L, respectively

% PRIMARY BIODEGRADATION
- 98.81, 98.87 and 99.18% at nominal concentrations of 1, 3, and 10 mg/L, respectively

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 51.3, 61.4 and 59.1% for 1, 3, and 10 mg/L, respectively

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: No data

The detailed results are provided in "Any other information on results incl. tables" section.
Results with reference substance:
No reference substance was run in the study.

Table 1: Results for degradation, sorption and mass balance for LAS study (WTDS# 42804)

Measured parameters (± SD) 1 mg/L 3 mg/L 10 mg/L
% Radiolabel removal based on LSC 89.3± 1.2 87.5 ± 3.1 89.0 ± 1.0
% Parent removal  99.44 ± 0.2 99.36 ± 0.2 99.37 ± 0.1
Estimated parent concentration in effluent (µg/L)  5.6 ± 1.7 19.2 ± 4.9 62.5 ± 13.6
% mineralization (14CO2) 51.3 ± 9.0 61.4 ± 2.9 59.1 ± 4.7
% primary biodegradation 98.81 98.87 99.18
Mean influent background concentration (mg/L) 0.48 0.36 0.74
Removal by sorption to MLSS (%) 1.9 2.2 1.8
LAS sorbed on MLSS (mg/kg)  90 ± 7 333 ± 16 503 ± 109
LSC Mass balance (%) 81.5± 7.5% 91.0± 6.5% 86.9± 3.5%

Table 2: Water quality analysis (TSS, COD, BOD, NO3, NH3 and PO4) (WTDS# 42804)

Measured parameter CAS influent Control effluent Test effluent 
Total suspended solids (TSS) (in mg/L) 174± 44 15± 11 13± 12
TSS removal NA 91.40% 92.50%
COD Analysis (in mg/L) 220± 32 26± 4 25± 7
COD removal NA 88.20% 88.60%
BOD5 Analysis (in mg/L) 93± 20  6± 0 Not detected
BOD5 removal NA 93.5%* 94.7%*
Total phosphate (PO4) analysis (in mg/L) 3.9± 0.68 2.66± 0.64 2.43± 0.58
PO4 removal NA 31.90% 37.60%
Ammonia (NH4) analysis (in mg/L) 13.76± 3.36 <2.0 <2.0
NH4 removal NA ≥85.5% ≥85.5%
Nitrate (NO4) analysis (in mg/L) 0.21± 0.24 14.4± 2.8 12.1± 2.3
Nitrate formation NA 98.50% 98.30%
pH analysis 7.9± 0.2 7.8± 0.1 (control MLSS)
8.0 ± 0.2(control effluent)		 7.7± 0.1 (test MLSS)
7.9 ± 0.2(test effluent)

*Since BOD was below detection limit, 4.9 mg/L was used to calculate removals

Table 3: Metabolites sorbed on MLSS as % of initial dose and concentration sorbed (WTDS# 42804)

Nominal dose Metabolite Metabolite (in dpm/mL) Metabolite (in %) Metabolite (in µg/L)
1 mg/L  LAS polar metabolite #1  75± 7 0.18 ± 0.01% 1.8 ± 0.1
1 mg/L  LAS polar metabolite #2  183 ± 7 0.44 ± 0.03% 4.4 ± 0.3
1 mg/L  LAS polar metabolite #3  61 ± 3 0.15 ± 0.00% 1.5 ± 0.0
1 mg/L LAS non-polar metabolite #1  40 ± 4 0.10 ± 0.01% 1.0 ± 0.1
1 mg/L LAS non-polar metabolite #2  153 ± 11 0.36 ± 0.03% 3.6 ± 0.3
3 mg/L  LAS polar metabolite #1  36 ± 7 0.08 ± 0.02% 2.5 ± 0.6
3 mg/L  LAS polar metabolite #2  97 ± 21 0.23 ± 0.06% 6.9 ± 1.7
3 mg/L  LAS polar metabolite #3  40 ± 3 0.09 ± 0.01% 2.8 ± 0.2
3 mg/L  LAS polar metabolite #4  72 ± 13 0.17 ± 0.03% 5.1 ± 1.0
3 mg/L  LAS polar metabolite #5  133 ± 14 0.31 ± 0.04% 9.3 ± 1.1
3 mg/L  LAS polar metabolite #6  86 ± 28 0.20 ± 0.07% 6.1 ± 2.1
3 mg/L LAS non-polar metabolite #1  50 ± 9 0.12 ± 0.02% 3.5 ± 0.6
3 mg/L LAS non-polar metabolite #2  234 ± 7 0.55 ± 0.13% 16.5 ± 3.8
10 mg/L  LAS polar metabolite #1  25 ± 3 0.06 ± 0.01% 5.6 ± 0.6
10 mg/L  LAS polar metabolite #2  81 ± 1.1 0.18 ± 0.03% 18.5 ± 3.2
10 mg/L  LAS polar metabolite #3  34 ± 5 0.08 ± 0.01% 7.8 ± 1.1
10 mg/L  LAS polar metabolite #4  58 ± 10 0.13 ± 0.02% 13.2 ± 2.2
10 mg/L  LAS polar metabolite #5  106 ± 19 0.24 ± 0.04% 24.3 ± 4.4
10 mg/L  LAS polar metabolite #6  64 ± 8 0.15 ± 0.02% 14.7 ± 1.8
10 mg/L  LAS polar metabolite #7  35 ± 9 0.08 ± 0.02% 8.1 ± 2.1
10 mg/L  LAS polar metabolite #8  48 ± 8 0.11 ± 0.02% 10.9 ± 1.7
10 mg/L  LAS non-polar metabolite #1  46 ± 7 0.11 ± 0.01% 10.5 ± 1.5
10 mg/L  LAS non-polar metabolite #2  197 ± 30 0.45 ± 0.07% 45.0 ± 6.6

Table 4: Metabolite in effluent as % of Dosed & effluent Concentration (Influent/ effluent) (WTDS# 42804)

Nominal dose Metabolite Metabolite (in dpm/mL) Metabolite (in %) Metabolite (in µg/L)
1 mg/L  LAS polar metabolite #1  8± 3 0.98 ± 0.43% 9.8 ± 4.3
3 mg/L  LAS polar metabolite #1  15 ± 5 1.85 ± 0.77% 55.6 ± 23.0
10 mg/L  LAS polar metabolite #1  16 ± 4 1.96 ± 0.43% 196.3 ± 42.7
1 mg/L  LAS polar metabolite #2 14 ± 7 1.74 ± 0.81% 17.4 ± 8.1
3 mg/L  LAS polar metabolite #2 7 ± 3 0.80 ± 0.34% 23.9 ± 10.2
10 mg/L  LAS polar metabolite #2 8 ± 3 0.96 ± 0.41% 95.9 ± 41.1
1 mg/L  LAS polar metabolite #3 26 ± 1 3.14 ± 0.03% 31.4 ± 0.3
3 mg/L  LAS polar metabolite #3 17 ± 6 2.04 ± 0.62% 61.1 ± 18.7
10 mg/L  LAS polar metabolite #3 17 ± 7 2.04 ± 0.77% 203.8 ± 76.5
1 mg/L  LAS polar metabolite #4 8 ± 3 1.00 ± 0.38% 10.0 ± 3.8
3 mg/L  LAS polar metabolite #4 19 ± 2 2.35 ± 0.41% 70.6 ± 12.3
10 mg/L  LAS polar metabolite #4 9 ± 3 1.07 ± 0.40% 106.8 ± 40.1
1 mg/L  LAS polar metabolite #5 0 0% 0
3 mg/L  LAS polar metabolite #5 0 0% 0
10 mg/L  LAS polar metabolite #5 9 ± 6 1.05 ± 0.67% 105.5 ± 66.9

Table 5: Mass balance based on combustion analysis for MLSS (WTDS# 42804)

Sample Mass balance (in %) Mean (in %)
1 mg/L, Day 1 118.7 117.9
1 mg/L, Day 5 116.9
3 mg/L, Day 1 174.6 165.4
3 mg/L, Day 5 156.3
10 mg/L, Day 1 105.2 110.3
10 mg/L, Day 5 115.4

Table 6: Results based on SAX cartridge verification (WTDS# 42804)

Nominal dose Parent in influent (dpm/mL) Parent in effluent (dpm/mL) Parent removal (%)
1 mg/L 784 ± 12 4.6 ± 1.3 99.4 ± 0.2%
3 mg/L 758.9 ± 35.3 5.4 ± 1.6 99.3 ± 0.2%
10 mg/L 838.9 ± 11.8 5.2 ± 1.1 99.4 ± 0.1%
Nominal dose Parent in influent (dpm/mL) Parent in effluent as % of dosed Parent in effluent (in µg/L)
1 mg/L 4.6 ± 1.3 0.56 ± 0.17% 5.6 ± 1.7
3 mg/L 5.4 ± 1.5 0.64 ± 0.16% 19.2 ± 4.9
10 mg/L 5.2 ± 1.1 0.63 ± 0.14% 62.5 ± 13.6
Validity criteria fulfilled:
yes
Conclusions:
In a continuous-flow activated sludge (CAS) study, linear alkylbenzenesulfonate was removed rapidly by 99.44, 99.36 and 99.37% at nominal concentrations of 1, 3 and 10 mg/L, respectively, in presence of domestic raw wastewater.
Executive summary:

A continuous-flow activated sludge (CAS) study was performed to evaluate the biodegradation potential of LAS by a method similar to OECD guideline 303 A. The focus of the study was to determine if effluent concentration was dependent or independent of influent concentration during secondary activated sludge wastewater treatment for linear alkyl benzenesulfonate (LAS). Two CAS systems (control unit and test unit) were operated at a hydraulic retention time (HRT) of 6 hours and a solids retention time (SRT) of 10-14 days. Domestic raw wastewater from Polk Run WWTP in Loveland, OH was used as a feed source for both CAS units. The control unit received only the domestic wastewater as a feed source. The testing unit was fed domestic wastewater along with 1, 3 and 10 mg/L concentrations of unlabeled sodium C12-C15 LAS and radiolabeled C14-C12 LAS (non-labeled LAS with a tracer of 14C radiolabeled LAS). Only one concentration was tested at a time with an equilibration period of approximately one month between testing concentrations. Initially a 2-phenyl isomer of 14C-C12LAS was tested at 1 mg/L mixed with a small amount of non-radiolabeled commercial mixture of LAS. All further testing was accomplished using a 3,4,5-phenyl isomer of 14C- C12LAS mixed with appropriate amounts of the non-labeled commercial LAS mixture. Mass balances were conducted for each system for adsorption to MLSS, mineralization, and overall system removal using liquid scintillation counting (LSC). Radio-thin layer chromatography (Rad-TLC) and combustion of mixed liquor suspended solids (MLSS) was used to determine the extent of parent and metabolite loss from the system, adsorbed onto, and incorporated into MLSS. LSC mass balance in all the tested concentration was in range of 80-100%. After 5 days, based on the radiolabelled LAS (14C-C12LAS) measurement, the % parent removal was 99.44, 99.36 and 99.37% for 1, 3, and 10 mg/L, respectively. Measured parent LAS effluent concentrations in the CAS effluent increased as nominal influent LAS concentrations were increased. The concentrations were 5.6, 19.2 and 62.5 µg/L. However, the observed increase in effluent concentrations were not fully proportional to the experimentally increased influent concentrations (1, 3, and 10 mg/L). The sum of effluent concentrations of parent plus metabolites increased proportionally as influent concentrations were increased. Mass of parent LAS and LAS intermediates, number of identified LAS polar intermediates sorbed to the CAS activated sludge mixed liquor solids were also increased as applied influent concentrations to the CAS systems were increased. No significant measured difference in removals were observed between 2-phenyl and 3,4,5-phenyl isomers tested. It was estimated that the residence time in the CAS (6-hours) was sufficient to remove both species of LAS to low levels in the final effluent. In a continuous-flow activated sludge (CAS) study, linear alkyl benzenesulfonate was removed rapidly by 99.44, 99.36 and 99.37% at nominal concentrations of 1, 3, and 10 mg/L, respectively, in presence of domestic raw wastewater. This biodegradation study is classified as acceptable and satisfies the guidelines requirement for the OECD 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units).

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
prior to 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Additional homologues were tested in the same study:
sodium (1-methylnonyl)benzenesulfonate
sodium (1-butylhexyl)benzenesulfonate
sodium (1-methylundecyl)benzenesulfonate
sodium (1-pentylheptyl)benzenesulfonate
sodium (1-methyltridecyl)benzenesulfonate
sodium decylbenzenesulfonate
sodium undecylbenzensulfonate
sodium dodecylbenzenesulfonate
sodium tetradecylbenzensulfonate
Radiolabelling:
yes
Remarks:
specific activity = 8.70 uCi/mmol (C13 mixed LAS)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
River water was collected over a 2 year period at Rapid Creek, a first-order stream in southwestern South Dakota. Rapid Creek is a tributary to the Cheyenne River. Water samples were collected 7 km downstream of Rapid Creek municipal wastewater treatment plant. The plant is a small trickling filter facility (~8 MM gal/day) of predominately domestic wastewater.
Details on source and properties of sediment:
Bottom sediments were collected over a 2 year period at Rapid Creek, a first-order stream in southwestern South Dakota. Rapid Creek is a tributary to the Cheyenne River. Water samples were collected 7 km downstream of Rapid Creek municipal wastewater treatment plant. The plant is a small trickling filter facility (~8 MM gal/day) of predominately domestic wastewater.
Details on inoculum:
1000 mg/L bottom sediments for river water/sediment flasks. 8-160 mg/L total suspended solids in river water only flasks.
Duration of test (contact time):
21 d
Initial conc.:
10 µg/L
Based on:
act. ingr.
Initial conc.:
100 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
TOC removal
Details on study design:
Biodegradation assays were done in a closed flow-through shake flask system. Duplicate or triplicate 2 L Erlenmyer flasks contained 1 L of river water, or 1 L of river water and 1000 mg/L bottom sediments. C14 LAS was added to the water at 5-500 ug/L concentrations, and to sediments at 100 ug/g. Test flasks were incubated at 24C and constantly agitated on a rotary platform shaker. The head space of the flasks were aerated with CO2-free air and 14CO2 was trapped in external base traps containing 100 mL of 1.5 N KOH. Periodically, 10 mL aliquots were taken from the flasks and filtered (2 um). Filters were washed with 5 mL deionized water or 50% ethanol. The filtrate was added to 125 mL biometer flasks and acidified with 1 mL of concentrated HCl. Acidification releases the 14CO2 present as carbonate or bicarbonate in solution and is then trapped in 2 mL of 1.5 N KOH present in the biometer flask side arm. 10 mL aliquots of the acidified filtrate, 1 mL aliquots from the flask side arms and the external base traps, and the washed filters were counted by standard liquid scintillation. 14CO2 production and complete mass balance of radiolabel at specific time points were determined. Correction factors were used to normalize all disintegrations per minute data to a 10 mL basis. Tests were terminated by adding 1 mL concentrated HCl to test flasks. After acidification and quantitation of nonvolatile and volatile radioactivity for mass balance, 50 mL aliquots from water and water/sediments were collected and extracted twice with ethyl ether (60 mL) and evaporated to dryness. The extracts were resuspended in scintillation cocktail and counted to determine radiolabel present with the same solubility as the parent LAS.
Reference substance:
not specified
Compartment:
natural water / sediment: freshwater
% Recovery:
100
St. dev.:
6
Remarks on result:
other: range of 96-112%
Key result
% Degr.:
> 70 - < 90
Parameter:
CO2 evolution
Remarks:
14CO2
Sampling time:
21 d
Key result
Compartment:
natural water: freshwater
DT50:
ca. 24 h
Type:
(pseudo-)first order (= half-life)
Temp.:
24 °C
Key result
Compartment:
natural sediment: freshwater
DT50:
ca. 20 h
Type:
(pseudo-)first order (= half-life)
Temp.:
24 °C
Mineralization rate (in CO2):
0.71 d-1
Transformation products:
not specified
Details on results:
Kinetics of biodegradation of LAS homologues in river water with high levels of sediment was comparable to the biodegradation kinetics of homologues and phenyl isomer in river water with low levels of suspended solids. Biodegradation kinetic measurements were not effected by the kinectics of sorption, since sorption equillibria was rapid (<3 hours). Rate constants for degradation of individual homologues showed little variation as a function of alkyl chainlength. The mean rate constant for degradation in water/sediment systems was 0.82/d, higher than in river water at 0.7/d, but not significantly different (P<0.05). The average half-life for mineralization of homologues in the presence of sediments was ~20, and in river water ~ 24 hours.

Table 2. Kinetic Parameters for Biodegradation of LAS Homologues and Phenyl Isomers in River Water

compound   conc, ug/L  k1, CO2 production/day  95% CI
 C10 LAS  10  0.71  0.54 -0.87
   100  0.53  0.42 -0.64
   10  0.82  0.59 -1.05
   100  0.93  0.75 -1.11
 C11 LAS  10  0.76  0.38 -1.13
   100  0.66  0.31 -1.00
 C12 LAS  10  0.68  0.16 -1.20
   100  0.77  0.17 -1.37
   10  0.81  0.13 -1.50
   100  0.79  0.36 -1.22
   10  0.61  0.26 -0.96
   100  0.62  0.30 -0.95
   5  0.60  0.26 -0.93
   50  0.77  0.29 -1.25
   500  0.74  0.29 -1.20
 C13 LAS  10  0.78  0.22 -1.33
   100  0.70  0.40 -1.00
 C14 LAS  10  0.61  0.32 -0.90
   100  0.62  0.44 -0.79
   10  0.50  0.33 -0.68
   100  0.62  0.27 -0.97
 0.70 +/- 0.11         
 C10 -2 -phenyl LAS  10  1.08  0.19 -1.98
   100  0.71  0.48 -0.94
 C10 -5 -phenyl LAS  10  0.67  0.26 -1.07
   100  0.90  0.75 -1.05
 C12 -2 -phenyl LAS  10  0.63  0.42 -0.83
   100  0.58  0.30 -0.86
 C12 -6 -phenyl LAS  10  0.50  0.22 -0.77
   100  (a)  
   10  0.53  0.11 -0.94
   100  0.66  0.17 -1.15
 C14 -2 -phenyl LAS  10  0.71  0.58 -0.84
   100  0.80  0.54 -1.06
 0.71 +/- 0.17(b)         

 (a) Not determined. (b) Mean k1 value, +/- standard deviation, across all experiments.

Table 3. Kinetic Parameters of Biodegradation of LAS Homologues in River Sediments

 compound(a)  amt sorbed, %(b)  k1, CO2, production/day  95% CI
 C10 LAS  5.2  0.89  0.21 -1.57
 C11 LAS  13.8  0.79  0.27 -1.31
 C12 LAS  31.3

 0.86

0.76

 0.67 -1.06

0.34 -1.18
 C13 LAS  62.7  0.89  0.17 -1.61
 C14 LAS  77.0  0.72  0.55 -0.89
 0.82 +/- 0.07(c)         

(a) Initial concentration of LAS homologue, 100 ug/g of dry sediment. (b) At equilibrium (<3h). (c) Mean k1 value, +/- standard deviation, across all experiments.

Validity criteria fulfilled:
not specified
Conclusions:
This study measured the kinetics of biodegradation of 10 C14 -ring-labeled LAS homologues and phenyl isomers in river water and sediments. The test materials covered the range of materials in commercial formulations. The mean rate constant for degradation in water/sediment systems was 0.82/d, higher than in river water at 0.7/d, but not significantly different (P<0.05). The average half-life for mineralization of homologues in the presence of sediments was ~20, and in river water ~ 24 hours.  Based on these results, mineralization of LAS homologues and phenyl isomers was extensive (~80%) and followed apparent first-order kinetics in river water/sediment systems. The rate and extent of mineralization were not significantly affected by alkyl chain length or phenyl position. Mineralization was not adversely affected by the presence of high levels of suspended sediments or competing homologues; biodegradation occured independently for different homologues.
Executive summary:

This study measured the kinetics of biodegradation of 10 14C-labeled LAS homologues and phenyl isomers in river water and sediments. The mean rate constant for degradation in water/sediment systems was 0.82/d, higher than in river water at 0.7/d, but not significantly different (P<0.05). The average half-life for mineralization of homologues in the presence of sediments was ~20, and in river water ~ 24 hours.  Based on these results, mineralization of LAS homologues and phenyl isomers was extensive (~80%) and followed apparent first-order kinetics in river water/sediment systems.

Description of key information

MEA-LAS dissociates in water to its constituents MEA and LAS. LAS is readily biodegradable in natural sediment and MEA is rapidly biodegraded in activated sludge and natural river water. Therefore, MEA-LAS is expected to be readily biodegradable.

Study 1 (C10-13 LAS):

The biodegradation of C10-13 LAS, sodium salt (average chain length 11.6) in sediment was evaluated in an aerobic die-away study conducted according to a method similar to OECD Guidelines 308 and 314. The inoculum (sediment) was collected from the Lytle Creek, Wilmington, Ohio. Radiolabelled test material (14C) was incubated with biotic and abiotic sediment samples under static conditions. The test material was added to the sediment to yield a final concentration of 1.5 mg/kg dry weight and the test system was incubated for 148 d. The evolved 14CO2 was analysed throughout the exposure duration. In addition, replicates of abiotic and biotic sediment were analysed by parent, metabolites and non–extractable radioactivity. The data collected from the abiotic samples was consistent and demonstrated that the parent remained stable throughout the test. In biotic samples, the parent LAS underwent primary degradation with the appearance of one non-polar metabolite (unidentified) with Rf 0.57. Test substance declined from 109% after 5 min to <27% by 61 d. Primary degradation was biphasic (two compartment first order model) with two pools of material exhibiting different degradation rates. The rates of primary degradation were 42.2 and 64.8% in Pools A and B, respectively. The rate constants for primary degradation were 1.5 and 0.007 d-1 and half-lives were 0.4 and 99 d in Pools A and B, respectively. The percent 14CO2 was 10.4% by Day 1 and increased to 60.8% by Day 148 for biotic samples. 14CO2 production was best described by a first order model, indicating that parent and metabolites were equally bioavailable to undergo mineralization. Based on above results, the test substance was aerobically biodegraded in sediment (Lytle Creek) at the end of 148 d exposure with about 60.8% being mineralized to CO2, 14.4% associated with solids, 1.4% as metabolites and 24.5% remaining as parent (Itrich, 2010a).

Study 2 (C10 -16 LAS):

A continuous-flow activated sludge (CAS) study was performed to evaluate the biodegradation potential of LAS by a method similar to OECD guideline 303 A. The focus of the study was to determine if effluent concentration was dependent or independent of influent concentration during secondary activated sludge wastewater treatment for linear alkyl benzenesulfonate (LAS). Two CAS systems (control unit and test unit) were operated at a hydraulic retention time (HRT) of 6 hours and a solids retention time (SRT) of 10-14 days. Domestic raw wastewater from Polk Run WWTP in Loveland, OH was used as a feed source for both CAS units. The control unit received only the domestic wastewater as a feed source. The testing unit was fed domestic wastewater along with 1, 3 and 10 mg/L concentrations of unlabeled sodium C12-C15 LAS and radiolabeled C14-C12 LAS (non-labeled LAS with a tracer of 14C radiolabeled LAS). Only one concentration was tested at a time with an equilibration period of approximately one month between testing concentrations. Initially a 2-phenyl isomer of 14C-C12LAS was tested at 1 mg/L mixed with a small amount of non-radiolabeled commercial mixture of LAS. All further testing was accomplished using a 3,4,5-phenyl isomer of 14C- C12LAS mixed with appropriate amounts of the non-labeled commercial LAS mixture. Mass balances were conducted for each system for adsorption to MLSS, mineralization, and overall system removal using liquid scintillation counting (LSC). Radio-thin layer chromatography (Rad-TLC) and combustion of mixed liquor suspended solids (MLSS) was used to determine the extent of parent and metabolite loss from the system, adsorbed onto, and incorporated into MLSS. LSC mass balance in all the tested concentration was in range of 80-100%. After 5 days, based on the radiolabelled LAS (14C-C12LAS) measurement, the % parent removal was 99.44, 99.36 and 99.37% for 1, 3, and 10 mg/L, respectively. Measured parent LAS effluent concentrations in the CAS effluent increased as nominal influent LAS concentrations were increased. The concentrations were 5.6, 19.2 and 62.5 µg/L. However, the observed increase in effluent concentrations were not fully proportional to the experimentally increased influent concentrations (1, 3, and 10 mg/L). The sum of effluent concentrations of parent plus metabolites increased proportionally as influent concentrations were increased. Mass of parent LAS and LAS intermediates, number of identified LAS polar intermediates sorbed to the CAS activated sludge mixed liquor solids were also increased as applied influent concentrations to the CAS systems were increased. No significant measured difference in removals were observed between 2-phenyl and 3,4,5-phenyl isomers tested. It was estimated that the residence time in the CAS (6-hours) was sufficient to remove both species of LAS to low levels in the final effluent. In a continuous-flow activated sludge (CAS) study, linear alkyl benzenesulfonate was removed rapidly by 99.44, 99.36 and 99.37% at nominal concentrations of 1, 3, and 10 mg/L, respectively, in presence of domestic raw wastewater. This biodegradation study is classified as acceptable and satisfies the guidelines requirement for the OECD 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units) (Kaiser, 1998).

Study 3 (C12 LAS):

This study measured the kinetics of biodegradation of 10 14C-labeled LAS homologues and phenyl isomers in river water and sediments. The mean rate constant for degradation in water/sediment systems was 0.82/d, higher than in river water at 0.7/d, but not significantly different (P<0.05). The average half-life for mineralization of homologues in the presence of sediments was ~20, and in river water ~ 24 hours.  Based on these results, mineralization of LAS homologues and phenyl isomers was extensive (~80%) and followed apparent first-order kinetics in river water/sediment systems (Larson, 1950).

Study 4 (C10-13 LAS):

The biodegradation of C10-13 LAS, sodium salt (average chain length 11.6) in sediment was evaluated in an aerobic die-away study conducted according to a method similar to OECD Guidelines 308 and 314. The inoculum (sediment) was collected from the Ohio River, Cincinnati, Ohio. Radiolabelled test material (14C) was incubated with biotic and abiotic sediment samples under static conditions. The test material was added to the sediment to a final added concentration of 1.5 mg/kg dry weight and the test system was incubated for 92 d. The evolved 14CO2 was analysed throughout the exposure duration. In addition, replicates of abiotic and biotic sediment were analysed by parent, metabolites and non-extractable radioactivity. The data collected from the abiotic samples was consistent and demonstrated that the parent remained stable throughout the test. In biotic samples, the parent LAS underwent primary degradation with the appearance of two non-polar metabolites (unidentified) with Rf 0.70 and 0.90. Test substance declined from 91% after 15 min to <22% by 92 d. Primary degradation was biphasic (two compartment first order model) with two pools of material exhibiting different degradation rates. The two pools were the same size but pool one was more readily bioavailable as indicated by a faster k1. The rates of primary degradation were 49.3 and 49.9% in Pools A and B, respectively. The rate constants for primary degradation were 0.5 and 0.009 d-1 and half-lives were 1.4 and 77 d in Pools A and B, respectively. The amount of 14CO2 was 6% by Day 1 and increased to 31.6% by Day 92 for biotic samples. 14CO2 production was best described by a first order model, indicating that parent and metabolites were equally bioavailable to undergo mineralization. At the end of 92 d exposure, about 31.6% was mineralized to CO2, 21.6% associated with solids and 22.6% remained as parent. The mass balance in this study was found to be 75.8% which was normalized to 100%. Based on 100% mass balance normalization, 42.1% was mineralized to CO2, 28.5% was associated with solids, 0% was metabolites and 29.8% remained as parent. Based on the above results, the test substance was aerobically biodegraded in sediment (Ohio River) at the end of 92 d exposure with about 42.1% being mineralized to CO2, 28.5% associated with solids and 29.8% remaining as parent (Itrich, 2010b).

Study 5 (MEA):

A Continuous Activated Sludge (CAS) study was conducted on monoethanolamine (MEA) according to OECD Guideline 303 A (Simulation Test - Activated Sludge Units). The test concentration was1.5 mg/L. The test material was a combination of radiolabelled and unlabelled MEA. Two sources of inoculum (activated sludge) were used:  from the Avondale Sewage Treatment Plant in Avondale, Pennsylvania, and from the Goose Creek Wastewater Treatment Plant in West Chester, Pennsylvania.  Wastewater was also collected from both sewage treatment plants. Four CAS units were used; a control and test unit received sewage wastewater and sludge either from the Avondale Plant or the Goose Creek Plant, respectively. Each CAS unit had a mixing chamber, aeration basin and cylindrical clarifier. Following stabilization (21 d) and acclimation period (11 d), the removal phase (12 d) was conducted. Samples (influent, aeration mixed liquor and effluent) were collected five times weekly during the removal phase and tested for the level of radioactivity present. The mean removal of MEA was 88% in the CAS study.  Removal was 88.1% (with Avondale sludge) and 88.4% (with Goose Creek sludge). No adverse effects on the overall CAS system performance were observed during any phases of the study period (Marks, 1994).

Study 6 (MEA):

A simulation of the mineralization of monoethanolamine (MEA) in activated sludge was conducted in accordance with the OECD Guideline 314B.  Radiolabeled MEA (14C) was tested at 1 mg/L. The inoculum was activated sludge (2500 mg/L TSS) obtained from a municipal sewage treatment plant that receives predominantly domestic waste (the Avondale STP). The test treatments were measured in triplicate. The cumulative percent of theoretical 14CO2 produced was 83.1% at Day 17 (mean of three flasks). The rate constant for mineralization of MEA in activated sludge was 0.04 d-1 (average of the 3 flasks, ranging from 0.03-0.07 d-1) (Marks, 1992).

Study 7 (MEA):

A simulation of the mineralization of monoethanolamine (MEA) in surface water was conducted under aerobic conditions in accordance with the OECD Guideline 314D. MEA was tested at 300 µg/L. Radiolabeled MEA was added at 0.45 µCi/L. The river water was collected from Brandywine Creek in Downingtown, Pennsylvania. The test treatments were measured in triplicate. Radiolabelled d-Glucose(14C) served as a reference control. After 50 d, the cumulative percent of theoretical 14CO2 produced was 86.9% (mean of flasks 1, 2 and 3). The mass balance was 94.5%. Normalized to 100% mass balance, mineralization of MEA at Day 50 was 91.8%. The rate constant for mineralization of MEA in surface water was 0.59 d‑1(mean of flasks 1, 2 and 3). This corresponds to a half-life of 1.06 d (Marks, 1993).

Key value for chemical safety assessment

Additional information