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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water and sediment: simulation tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25 Aug 1992 - 22 Apr 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314B. Deviations, reliability, and validity evaluated against current OECD 314B (Oct. 3, 2008)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314A. Deviations, reliability, and validity evaluated against current OECD 314A (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA GLP
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: sewage influent, or activated sludge
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: Source of activated sludge and raw sewage (influent) was Sycamore Sewage Treatment Plant (STP) located in Loveland, Ohio. This STP receives predominantly domestic waste. The samples were collected on three different dates for the following experiments:
Experiment 1: Inoculum was raw sewage collected on Sept. 14, 1992
Experiment 2: Inoculum was activated sludge collected on Aug. 25, 1992
Experiment 3: Inoculum was activated sludge collected on Oct. 09, 1992
- Storage conditions: Activated sludge was aerated until use.
- Storage length: Not reported
-Concentration of sludge:
Experiment 1: Not reported
Experiment 2: 2670 mg/L
Experiment 3: 2426 mg/L
Duration of test (contact time):
24 h
Initial conc.:
0.5 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
other: metabolites... (see attached file)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 250-500 mL
- Composition of medium: Each test beaker contained
250-500 mL inoculum
0.5 mg/L test material
- Test temperature: Not reported
-pH adjusted: Yes. The pH of both abiotic and bioactive sludge was adjusted to approx. 7.0 before initiating the experiments.
- Aeration of dilution water: Not specified in the study report.
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: Beakers containing influent sewage/sludge and test chemical
- Number of culture flasks/concentration: One
- Method used to create aerobic conditions: Not reported
- Measuring equipment: CO2 measuring apparatus is not reported in the study and mineralization to 14CO2 was determined indirectly by measuring the difference in total radioactivity between samples from the biotic and abiotic treatments.
- Details of trap for CO2 and volatile organics if used: Not used.

SAMPLING
- Sampling frequency:
Experiment 1 and 2: At 0, 5, 15, 30, 45, 60, 90, 120, 180 min and 24 h.
Experiment 3: At 0, 5, 15, 30, 60, 90, 120, 210 min; 4.5 and 24 h.
- Sampling method: At each sampling time, the following samples were collected:
For analysis of mineralization: 1 mL (2-5 replicates) mixed liquor samples were removed from each test flask and acidified with 10% HCL and allowed to sit overnight.
For analysis of parent compound and metabolite: 3-10 mL sample was removed from each test flask and immediately flash frozen in a dry ice-acetone bath. These frozen samples were then lyophilized.
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes (Sludge was autoclaved for 90 min. and amended with mercuric chloride (1g/L, pH 7) to serve as an abiotic control)
Toxicity control: No

STATISTICAL METHODS: Not reported
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
100
% Degr.:
0
Parameter:
CO2 evolution
Remarks:
(mineralization)
Sampling time:
24 h
Remarks on result:
other: Exp. 1 (Inoculum: Raw sewage)
% Degr.:
1.2
Parameter:
other: % of radioactivity as parent
Sampling time:
24 h
Remarks on result:
other: Exp. 1 (Inoculum: Raw sewage)
% Degr.:
65.71
Parameter:
other: % of radioactivity as metabolite
Sampling time:
24 h
Remarks on result:
other: Exp. 1 (Inoculum: Raw sewage)
% Degr.:
65.52
Parameter:
CO2 evolution
Remarks:
(mineralization)
Sampling time:
24 h
Remarks on result:
other: Exp. 2 (Inoculum: Activated sludge)
% Degr.:
0.85
Parameter:
other: % of radioactivity as parent
Sampling time:
3 h
Remarks on result:
other: Exp. 2 (Inoculum: Activated sludge). Not enough dpms to determine value for 24 h
% Degr.:
7.27
Parameter:
other: % of radioactivity as metabolite
Sampling time:
3 h
Remarks on result:
other: Exp. 2 (Inoculum: Activated sludge). Not enough dpms to determine value for 24 h
% Degr.:
51.41
Parameter:
CO2 evolution
Remarks:
(mineralization)
Sampling time:
24 h
Remarks on result:
other: Exp. 3 (Inoculum: Activated sludge)
% Degr.:
3.34
Parameter:
other: % of radioactivity as parent
Sampling time:
4.5 h
Remarks on result:
other: Exp. 3 (Inoculum: Activated sludge). Not enough dpms to determine value for 24 h
% Degr.:
16.11
Parameter:
other: % of radioactivity as metabolite
Sampling time:
4.5 h
Remarks on result:
other: Exp. 3 (Inoculum: Activated sludge). Not enough dpms to determine value for 24 h
Compartment:
other: activated sludge
DT50:
2 min
Type:
other: 3/2 order model
Remarks on result:
other: Half-life of primary biodegradation (Exp. 2)
Compartment:
other: activated sludge
DT50:
21.2 min
Type:
other: 3/2 order model
Remarks on result:
other: Half-life of mineralization (Exp. 2)
Compartment:
other: activated sludge
DT50:
2.4 min
Type:
other: First order
Remarks on result:
other: Half-life of primary biodegradation (Exp. 3)
Compartment:
other: activated sludge
DT50:
3.8 h
Type:
other: First order
Remarks on result:
other: Half-life of mineralization (Exp. 3)
Other kinetic parameters:
first order rate constant
other: 3/2 order rate constant (provided the best statistical fit) 0.42±0.014 min-1 for primary degradation (Exp. 2) ... (see attached file)
No.:
#1
Details on transformation products:
Not reported
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

MAJOR TRANSFORMATION PRODUCTS: In all the three experiments, the predominant metabolite was a polar substance, presumptively identified as PEG-sulfate.

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

EXTRACTABLE RESIDUES
- % of applied amount at Day 0:
Exp. 1: Active flask = 40.74%, Abiotic flask = 88.25% (parent and metabolite; sample time was 0 min)
Exp. 2: Active flask = 72.64%, Abiotic flask = 47.95% (parent and metabolite; sample time was 0 min)
Exp. 3: Active flask = 87.95%, Abiotic flask = 91.37% (parent and metabolite; sample time was 0 min)

- % of applied amount at end of study period:
Exp.1: Active flask = 66.91%, Abiotic flask = 89.04% (parent and metabolite; sample time was 24 h)
Exp. 2: Active flask = 8.12%, Abiotic flask = 97.58% (parent and metabolite; sample time was 180 min)
Exp. 3: Active flask = 19.45%, Abiotic flask = 86.4% (parent and metabolite; sample time was 4.5 h)
Enough dpms were not observed to determine value for 24 h in Exp. 2 and 3

NON-EXTRACTABLE RESIDUES:
– % of applied amount at Day 0:
Exp. 1: Active flask = 0%, Abiotic flask = 11.75% (sample time was 0 min)
Exp. 2: Active flask = 21.47%, Abiotic flask = 37.81% (sample time was 0 min)
Exp. 3: Active flask = 12.04%, Abiotic flask = 0% (sample time was 0 min)

- % of applied amount at end of study period:
Exp. 1: Active flask = 33.09%, Abiotic flask =10.96 % (sample time was 24 h)
Exp. 2: Active flask = 29.71%, Abiotic flask = 0% (sample time was 24 h)
Exp. 3: Active flask = 49.3%, Abiotic flask = 13.6% (sample time was 4.5 h)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study (24 hours): 65.52 (Exp. 2) and 51.41% (Exp. 3) in activated sludge bioactive test flasks; 0 in raw sewage bioactive test flask (Exp. 1). CO2 evolution in abiotic flask was 0% in exp. 1 and 3 while 4.87% in Exp. 2 for abiotic flask as radioactivity in the abiotic control was recovered primarily as intact parent.
For details, please refer to ‘table 1’ and ‘table2’ of all the three experiments in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION: No volatilization of test material was observed as the TLC analysis of the abiotic control revealed that the parent test material remained intact in all the three experiments.

Other: Characterization of AES metabolite: Non-diluted activated sludge (collected from Sycamore STP) and a very high concentration (250 mg) of AES was incubated. The intermediate generated was subsequently characterized based upon its behavior during solid phase extraction (SPE) using a variety of sorbent materials including C18, a strong cation exchange resin (SCX), a weak anion exchange resin (NH2) and a strong anion exchange resin (SAX). The intermediate was not retained on the CIS or SCX column, indicating that it was polar but not cationic. It was retained on the weak and strong anion exchange resins at pH 2 and 7, indicating that it remains anionic at acidic pH. This behavior is consistent with PEG-sulfate (metabolite)


Results with reference substance:
Not applicable

Experiment 1: Aerobic die-away of Alkyl Ethoxylate Sulfate in Raw sewage (Study # 35531)

Table 1: Biotic flask

time (min)

Percent of Initial Radioactivity Recovered As:

Mass balance

CO2

Polar metabolite (Rf 0.06)

Parent (Rf 0.46)

Non polar metabolite (Rf 0.91)

Non- extracted

0

0

0

40.74

0

0

40.74

5

0

1.58

91.1

0.3

7.03

100.01

15

0

3.03

83.66

0

13.31

100

30

0

3.63

74.63

0.18

21.57

100.01

45

0

5.07

79.93

0.13

14.87

100

60

0

8.1

74.3

0

17.6

100

90

0

9.59

67.07

0.28

23.06

100

120

10.23

10.39

68.94

0.27

10.17

100

180

0

16.74

68.45

0.79

14.02

100

24 h

0

65.71

1.2

0

33.09

100

 

 

Table 2 (Abiotic flask)

time (min)

Percent of Initial Radioactivity Recovered As:

Mass balance

CO2

Polar metabolite (Rf 0.06)

Parent (Rf 0.46)

Non polar metabolite (Rf 0.91)

Non- extracted

0

0

0

88.25

0

11.75

100

5

0

0

83.49

0

16.51

100

15

0

0

87.55

0

12.45

100

30

0

0

83.5

0

16.5

100

45

0

0

88.56

0

16.44

105

60

0

0

85.01

0

14.99

100

90

0

0

72.83

0

27.17

100

120

0

0

76.86

0

23.14

100

180

0

0

60.53

0

39.47

100

24 h

0

0

89.04

0

10.96

100

 

Experiment 2: Aerobic die-away of Alkyl Ethoxylate Sulfate in activated sludge collected on 25 Aug, 1992 (Study # 35531)

Table 1: Biotic flask

Time (min)

Percent of Initial Radioactivity Recovered As:

 

CO2

Polar   Rf 0.14

Parent   Rf 0.45

Non-Polar Rf 0.6

Non-extracted

Mass balance

0

5.88

11.99

60.65

0

21.47

99.99

5

19.88

34.96

7.59

7.05

30.53

100.01

15

22.37

29.01

1.41

3.46

43.74

99.99

30

28.27

21.48

0.29

4

45.96

100

45

37.45

11.99

0.63

2.14

47.79

100

60

40.91

11.58

0.49

1.37

45.62

99.97

90

45.91

9.65

0.69

1.27

42.48

100

120

45.23

9.61

0.88

1.45

42.87

100.04

180

48.05

6.03

0.85

1.24

43.82

99.99

24h

65.52

not enough dpms to determine

 

29.71

 

 

 

Table 2 (Abiotic flask)

Time (min)

Percent of Initial Radioactivity Recovered As:

 

 

CO2

Polar   Rf 0.14

Parent   Rf 0.45

Non-Polar Rf 0.6

Non-extracted

Mass Balance

0

14.24

0

47.95

0

37.81

100

5

0

0

82.04

0

17.96

100

15

0

0

85.98

0

14.02

100

30

0

0

82.01

0

17.99

100

45

0

0

89.24

0

10.76

100

60

0.37

0

84.43

0

15.2

100

90

3.8

0

82.79

0

13.41

100

120

7.86

0

97.58

0

0

105.44

180

4.87

0

97.58

0

0

102.45

 

Experiment 3: Aerobic die-away of Alkyl Ethoxylate Sulfate in activated sludge collected on 9 Oct, 1992 (Study # 35531)

Table 1: Biotic flask

Time (min)

Percent of Initial Radioactivity Recovered As:

CO2

Polar #1 0.1 

Polar #2 0.28

Parent   Rf 0.58

Non-extracted

Mass balance

0

0.00

0.00

20.19

67.76

12.04

99.99

5

1.09

2.84

49.37

21.43

25.27

100.00

15

-3.27

4.87

64.02

3.66

30.72

100.00

30

5.85

4.90

61.15

2.45

25.65

100.00

60

8.39

4.41

54.47

1.87

30.86

100.00

90

7.77

3.36

46.56

2.51

39.81

100.01

120

14.21

3.16

42.90

2.29

37.43

99.99

210

19.53

2.71

23.21

3.37

51.18

100.00

4.5 h

30.64

1.98

14.13

3.34

49.30

99.39

24h

51.41

not enough dpms to determine

 

37.00

 

Table 2 (Abiotic flask)

Time (min)

Percent of Initial Radioactivity Recovered As:

CO2

Polar #1 0.1 

Polar #2 0.28

Parent   Rf 0.58

Non-extracted

Mass balance

0

25.88

0.00

0.00

91.37

0.00

117.25

5

16.53

0.00

0.00

94.35

0.00

110.88

15

4.95

0.00

0.00

88.98

6.06

99.99

30

4.70

0.00

0.00

90.12

5.18

100.00

60

0.00

0.00

0.00

89.46

10.54

100.00

90

0.00

0.00

0.00

89.34

10.66

100.00

120

0.00

0.00

0.00

89.83

10.17

100.00

210

0.00

0.00

0.00

91.64

8.36

100.00

4.5 h

0.00

0.00

0.00

86.40

13.60

100.00

Validity criteria fulfilled:
yes
Conclusions:
The removal of C14E3S (alkyl ethoxylate sulfate, AES) was 98.8% in raw sewage (24 hr die away test), and 98.0% in activated sludge (mean of two <5 hr die away tests). In the raw sewage die away, AES was converted to metabolite(s) and incorporated into solids. In the active sludge die aways, AES was mineralized, coverted to metabolites, and incorporated into solids.
Executive summary:

The biodegradation of C14E3S (alkylethoxysulfate, AES) was evaluated in three aerobic die-away studies, conducted in accordance with the OECD 314A and 314B guidelines. The aerobic die-away tests either used raw sewage or activated sludge (collected from Sycamore STP on different dates) as inoculum. This municipal wastewater treatment plant receives predominantly domestic waste. A solution of radiolabeled alkyl ethoxylate sulfate was tested at 0.5 mg/L.

The removal of AES in activated sludge was evaluated in 2 die-away experiments. In experiment #2, 99.2% of the AES was removed in 3 hr. Removal at 24 hr could not be calculated because of insufficient dpms to quantify the amount of metabolite and parent at 24 hr. Removal at 24 hr, based only on mineralization and incorporation into solids, was 95.2%. Rate constants for removal were 0.42 min-1 (primary biodegradation) and 0.033 min-1 (mineralization).

 

In experiment #3, 96.7% of the AES was removed in 4.5 hr. Removal at 24 hr could not be calculated because of insufficient dpms to quantify the amount of metabolite and parent at 24 hr. Removal at 24 hr, based only on mineralization and incorporation into solids, was 88.4%. Rate constants for removal were 0.29 min-1 (primary biodegradation) and 0.003 min-1 (mineralization).

 

The removal of AES from raw sewage was 98.8% (24 hr die-away, experiment #1). After 24 hr, 65.7% of the AES was converted to metabolite, and 33.1% was incorporated into solids. Rate constants are not available in the study report. After 3 hr in raw sewage, removal of AES was 31.6%.

 

The results from this experiment can be summarized as follows:

Removal of AES in Raw Sewage and Activated Sludge (study # 35531)

Die-Away in:

Removal of Parent (%)

Rate constant (primary biodegradation)

Rate constant (mineralization)

Activated Sludge*

98.0*

0.36 min-1

0.018 min-1

Raw Sewage

98.8

---

---

* Mean of 2 tests.  Removal at 3-4.5 hrs. All other results are at 24 hrs.

 

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

Endpoint:
biodegradation in water and sediment: simulation tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 Mar 18 Jul 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314D. Deviations, reliability, and validity evaluated against current OECD 314D (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA GLP
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: Mixture of Surface water, sewage effluent and activated sludge
Details on source and properties of surface water:
- Details on collection: The river water was collected from the Lower East Fork of the Little Miami River (Milford, OH) above the outfall of the Lower East Fork Sewage Treatment Plant (STP). The sample was collected on April 4, 1994
- Storage conditions: The water was aerated until use
- Storage length: Not specified
Details on source and properties of sediment:
Not applicable
Details on inoculum:
- Source of inoculum/activated sludge: The effluent was collected from the lower East Fork STP (Milford Ohio), and the activated sludge was collected from Sycamore Sewage Treatment Plant (Cincinnati, OH). The samples were collected on April 4, 1994.
Duration of test (contact time):
31 d
Initial conc.:
75 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
other: biomass... (see attached file)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 660 µL dosing solution was used for each flask
- Additional substrate: None
- Solubilising agent: Not used
- Test temperature: The test bottles were incubated at 22°C and gently mixed on shaker table.
- pH: The pH of biotic and abiotic flasks was 8.2 prior to dosing the test material
- Aeration of dilution water: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L Erlenmeyer flask consisted of 1.4 L of a river water mixture (89% river water, 10% sewage effluent, 1% activated sludge supernatant) and 660 µL dosing solution
- Number of culture flasks/concentration: One
- Method used to create aerobic conditions: The headspace of the test flasks were continuously purged with CO2 free air.
- Measuring equipment: Carbon dioxide measuring apparatus consisted of: (a) CO2 scrubber: i) Ascarite trap and ii) Three methyl red indicating traps to serve as a CO2 indicator trap. These traps connected in series with to a pressurized air source (b) CO2 production apparatus: 2 L Erlenmeyer flasks connected by tubing to the CO2-free air source; (c) Three CO2 absorber bottles: Absorber bottles filled with 1.5 N KOH
- Test performed in closed vessels: Yes
- Details of trap for CO2 and volatile organics: Bottles containing 1.5 N KOH were used as base traps for CO2
- Other: To terminate the experiment, the test flasks were acidified with 5 mL of concentrated HCI, injected through a sampling port. The flasks were purged with CO2 free air for several days and the base traps were sampled a final time.

SAMPLING
- Sampling frequency: At 1, 15, 30 min; at 1, 2, 4, 6, 9.25, 13.25, 17.25, 22.5, 30 hours; and at 2, 3, 6, 7, 8, 10, 14, 21, 28, and 31 days (after acidification).
- Sampling method: At each sampling time, the following samples were collected:
For analysis of Evolved 14CO2: The first KOH trap in the series was opened and sampled. Triplicate 1 mL aliquots were removed and mixed with 15 mLUltima Gold scintillation cocktail. This trap was reconnected or removed, in which case the traps were moved forward and a fresh trap added behind the existing traps.
For analysis of Dissolved CO2: A 7 mL sample was removed and the pH adjusted to approx. 10 with 200 µL of 1.5 N KOH. Six 1mL aliquots were placedin separate microcentrifuge tubes, three of these samples were centrifuged and the supernatant was collected for further analysis. The remaining three tubes were centrifuged, the supernatant was transferred to scintillation vials and acidified with 250 µL of 10% HCI.
For analysis of parent compound, metabolite and radioactivity associated with solids: 15 mL sample was removed from each test flask and immediately flash frozen in a dry ice-acetone bath.
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes (River water was autoclaved and amended with mercuric chloride (1g/L) to serve as an abiotic control)
- Toxicity control: No

STATISTICAL METHODS: The loss of parent (%) data were fitted to various decay equations and the mineralization (%CO2) and complete biodegradation (% CO2 +% biomass) data were fitted to production equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D (version 2.00) software. The equation that provided the best fit were identified based upon statistical considerations (r2 and number of parameters estimates) and visual inspection of the fit and residuals
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
98.3
St. dev.:
4
% Degr.:
39.7
Parameter:
CO2 evolution
Remarks:
(Mineralization)
Sampling time:
48 h
Remarks on result:
other: Normalized to 100% mass balance.
% Degr.:
60.3
Parameter:
other: % of radioactivity associated with solids
Sampling time:
48 h
Remarks on result:
other: Normalized to 100% mass balance.
% Degr.:
0
Parameter:
other: % of radioactivity as parent and metabolite
Sampling time:
48 h
Compartment:
water
DT50:
0.71 h
Type:
other: First order
Remarks on result:
other: Half-life of primary biodegradation
Compartment:
water
DT50:
11.4 h
Type:
other: First order
Remarks on result:
other: Half-life of complete biodegradation
Other kinetic parameters:
first order rate constant
other: 0.028 ±0.008 h-1 for mineralization (3/2 order rate constant without growth provided the best statistical fit)
Transformation products:
not measured
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
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

EXTRACTABLE RESIDUES: TLC analysis of the bioactive samples revealed the presence of 4 peaks with the Rf values of 0, 0.10, 0.23, and 0.56. Most abiotic samples exhibited a single peak at Rf 0.56 which corresponded to the parent alkyl ethoxy sulfate, which was also observed in early bioactive samples
- % of applied amount at Day 0: 73.4% in active flask (parent; sample time was 0.08 h)
- % of applied amount at end of study period: 0% in active flask (parent; sample time was 192 h)

NON-EXTRACTABLE RESIDUES: Incorporation into biomass was very rapid and reached a maximum of 53% after 48 h and subsequently plateaued at approx. 30% for the remainder of the study.
- % of applied amount at Day 0: 19.8 % in active flask (sample time was 0.08 h)
- % of applied amount at end of study period: 38.3% in active flask (sample time was 192h)

MINERALISATION: Following a lag period (13 h), 14CO2 evolution equaled 47% after 3 d and 72% after 21 d.
- % of applied radioactivity present as CO2 at end of study: 60.7% in bioactive test flask (sample time was 192 h). CO2 evolution was not analyzed for abiotic flask as radioactivity in the abiotic control was recovered primarily as intact parent. For details, please refer to ‘table 1’ and ‘table 2’ in the ‘Any other information on results incl. tables’ section.

PARENT
- % of the applied radioactivity present remaining as parent at end of study: The disappearance of parent alkyl ethoxy sulfate from the test system was rapid, and no parent was detected after 6 h.

Mineralization test of Alkyl Ethoxy sulfate (study # 35563)

Table 1: Bioactive flask

Time (h)

%Parent

%CO2

%Metabolites

Total % biomass

* Total biodegradation

Mass balance

0.08

73.4

NA

6.7

19.8

NA

99.9

0.26

51.8

2.9

27.6

17.8

20.7

100.1

1

36.4

5.6

28.2

29.8

35.4

100

2

13.4

0.6

63.1

22.9

23.5

100

4

2.3

4.1

44.4

49.2

53.3

100

9.25

0

1.9

56.3

41.8

43.7

100

13.25

0

2.1

46.4

51.5

53.6

100

22.5

0

29.2

31.5

39.4

68.6

100.1

30

0

34

16.7

49.5

83.5

100.2

48

0

34.6

0

52.5

87.1

87.1

72.5

0

46.6

0

46.8

93.4

93.4

192

0

60.7

0

38.3

99

99

Mean

98.3±4.0

Abiotic control (mean± S.D)

92.3±2.6

NA

0.56±1.6

5.5±1.8

NA

 

* CO2 +incorporation into biomass.

The specific AES compound tested was C14E3S.

Table 2: Abiotic flask

Sample Time (h)

% Parent

%CO2

% Metabolite

Total % biomass

Mass balance

0.25

91.7

NA

0

8.3

100

9.25

93.9

NA

0

6.1

100

48

93.2

NA

0

6.8

100

Validity criteria fulfilled:
yes
Conclusions:
C14E3S (Alkyl ethoxy sulfate, AES) was completely removed from surface water. After 48 h, 39.7% was mineralized, 60.3% was non-extractable (solids), and 0% remained as either parent compound or metabolite. The rate constants for primary biodegradation, ultimate biodegradation, and mineralization in surface water were 0.97, 0.061, and 0.028 h-1, respectively.
Executive summary:

A simulation of the biodegradation of Alkyl ethoxy sulfate in surface water was conducted under aerobic conditions in accordance with the OECD 314D guideline. The surface water in this test was 89% river water, 10% sewage treatment plant effluent, and 1% activated sludge supernatant. A solution of radiolabeled C14E3S (Alkyl ethoxy sulfate, AES) was tested at 75 µg/L. The inoculum was river water, supplemented with a small portion of effluent and activated sludge). The river water was obtained from the East Fork of the Little Miami River above the outfall of the Lower East Fork Sewage Treatment Plant (STP), which was the source of the effluent.  Activated sludge was collected from the Sycamore Sewage Treatment Plant.

After 48 h, 39.7% was mineralized, 60.3% was non-extractable (solids), and 0% remained as parent or metabolite. 

The rate constants for biodegradation of alkyl ethoxy sulfate in surface water were:

Primary biodegradation: 0.97 h-1

Ultimate biodegradation: 0.061 h-1

Mineralization: 0.028 h-1

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

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Jul - 10 Aug 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314C. Deviations, reliability, and validity evaluated against current OECD 314C (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA GLP
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Inoculum or test system:
digested sludge
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: Anaerobic digester sludge was collected from Glendale Wastewater Treatment Facility (Cincinnati, OH). This waste water treatment facility receives predominantly domestic waste.
- Storage conditions: The sludge was transferred to an anaerobic chamber (Forma Scientific, Model 1024) containing oxygen-free nitrogen.
- Storage length: Not specified
Duration of test (contact time):
14 d
Initial conc.:
1 mg/L
Based on:
act. ingr.
Initial conc.:
10 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CH4 evolution
CO2 evolution
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 300 mL
- Composition of medium: Each 500 mL glass bottle contained: 300 mL sludge mixture (sludge was mixed with an equal volume of mineral salts solution), and 1 or 10 mg/L of radiolabelled test material
- Test temperature: 35°C
- pH: Not reported
- Aeration of dilution water: No. The test was conducted under anaerobic condition.
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 500 mL glass bottles
- Number of culture flasks/concentration: Two test bottles per concentration
- Method used to create anaerobic conditions: The test bottles were purged continuously with nitrogen at a rate of approx. 5 mL/min
- Measuring equipment: To measure the evolution of 14CO2 and 14CH4, the headspace of the bottles was continuously purged with nitrogen which was then passed through two CO2 traps containing 100 mL 1.5 N KOH. The effluent gas was then channeled through combustion tube (a quartz column packed with cupric oxide at 800 °C) to combust methane to carbon dioxide. The combustion process was aided with the addition of oxygen (approx. 2 mL/min) to the combustion tube. The combustion process converted CH4 to CO2 which was subsequently captured in another series of KOH traps.
- Test performed in closed vessels: Yes
- Details of trap for CO2 and volatile organics if used: Bottles containing 100 mL 1.5 N KOH were used for trapping radioactive gases. Each sample vessel has two trapping trains, one for CO2 trapping immediately following the reactor vessels and one for CH4 trapping, following the combustion apparatus.
- Other: To terminate the experiment, the test systems were acidified with 10 mL 5N H2SO4 by injected through a sampling port and nitrogen purging was continued. One final set of base trap samples was collected and analyzed next day.

SAMPLING
- Sampling frequency: At Day 0, 1, 2, 5, 9 and 14
- Sampling method: The first base trap in the trapping train was removed. The remaining 2 traps were moved forward, and a new trap added behind the existing traps
- Sample storage before analysis: Not specified
CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes (sludge was autoclaved and amended with mercuric chloride (1g/L) to serve as an abiotic control)
- Toxicity control: No

STATISTICAL METHODS: Not reported
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
95.7
% Degr.:
88.4
St. dev.:
1.3
Parameter:
other: 14CO2and 14CH4 evolution
Sampling time:
14 d
Remarks on result:
other: At conc. 1 mg/L (Mean of replicates)
% Degr.:
87.6
St. dev.:
0.3
Parameter:
other: 14CO2and 14CH4 evolution
Sampling time:
14 d
Remarks on result:
other: At conc. 10 mg/L (Mean of replicates)
Compartment:
water
DT50:
0.67 d
Type:
other: First order
Remarks on result:
other: Half-life of mineralization
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
yes
Details on results:
TEST CONDITIONS
- Anaerobicity, 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 or CH4 at end of study: 88.4 and 87.6% at 1 and 10 mg/L test material treatment, respectively. No lag period was observed with either concentration. The ratio of carbon dioxide to methane was 1:1.7 for the 1 mg/L treatment and 1:2 for the 10 mg/Ltreatment.

Table1: Mineralization test of Alkyl ethoxylate sulfate (AES) (study # 35548)

Treatment

% Recovered as 14CO2

% Recovered as 14CH4

Total gas recovered

% Remaining in solution

% Remaining with solids

Mass balance

14AES (1 mg/L)

Replicate 1

33.2

54.3

87.5

2.5

-

-

replicate 2

32.8

56.5

89.3

2.8

-

-

mean±s.d

33.0±0.2

55.4± 1.5

88.4± 1.3

2.6 ± 0.2

4.6

95.6

14AES (10 mg/L)

Replicate 1

29.3

58.5

87.8

2.5

-

-

replicate 2

29.3

58.2

87.5

2.7

-

-

mean±s.d

29.3±0.0

58.3 ±0.2

87.6 ± 0.3

2.6±0.1

5.7

95.9

The test material was C14E3S (AES).

Validity criteria fulfilled:
yes
Conclusions:
Alkyl ethoxylate Sulfate (C14E3S) underwent rapid and extensive mineralization in anaerobic digester sludge. At Day 14, the mineralization (CO2 + CH4) was 88% in both treatments (1 and 10 mg/L). Half-lives for mineralization of both treatments were 0.67 d.
Executive summary:

A simulation of the mineralization of Alkyl ethoxy sulfate (AES) in anaerobic digester sludge was conducted in accordance with OECD 314C guideline. Radiolabeled alkyl ethoxy sulfate (14C-C14E3S) was tested at 1 and 10 mg/L. The inoculum was anaerobic digester sludge collected from Glendale Wastewater Treatment Facility. This treatment facility receives predominantly domestic waste. The test treatments were measured in duplicate.

After 14 d, 88% of both concentrations of 14C-C14E3S were recovered as 14C-gases.

Half-lives for mineralization of both treatments were 0.67 d.

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

Endpoint:
biodegradation in water and sediment: simulation tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 Sep 1993 - 04 Mar 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Followed guideline, well documented, with GLP
Qualifier:
equivalent or similar to
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:
other: Mixture of river water and domestic activated sludge.
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 Sept. 22, 1993 using plastic containers.
- Storage conditions: Water samples were composited and stored under refrigeration.
- Storage length: 1 d
- Temperature (°C) at time of collection: Not reported
- pH at time of collection: 6.7
- Oxygen concentration (mg/l) initial/final: 4.4 mg/L
- Hardness (CaCO3): 119 mg/L
- Total organic carbon (%): 4.4 mg/L
- Biomass: 1.1 x 10(3) CFU/mL (river water); 1.4 x 10(3) CFU/mL (river water supplemented with inoculum)
- TSS: 3 mg/L
Details on source and properties of sediment:
Not applicable
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from Downingtown Regional Water Pollution Control Center, Downingtown, Pennsylvania. This sewage treatment plant receives predominantly domestic waste.
- Storage conditions: Activated sludge was held overnight with aeration.
- Storage length: Overnight
Preparation of inoculum for exposure: After receiving, approx. 300 mL of the activated sludge was composited and homogenized at medium speed ina blender for approx. 2 min. The homogenized sample was poured into a beaker and allowed to settle for approx. 30 min. The clear supernatant is decanted to provide 0.1% (v/v) inoculum in each CO2 test bottle.
- Pretreatment: None
- Concentration of sludge: The sludge was adjusted to a target total suspended solids level of 2500 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 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 river water
- Composition of medium: Each 2 L test flask contained:
1 L river water
1 mL activated sludge
100 µg active/L dosing solution

- Test temperature: 21.4- 23.5 °C
- pH: Not reported
- Aeration of dilution water: Yes, please see "Method used to create aerobic conditions" below.
- Suspended solids concentration: 2500 mg/L (the activated sludge)
- 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 flasks
- Number of culture flasks/concentration: Duplicate
- 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 purged through the scrubbing train at a constant rate which is adequate to provide 1-2 bubbles/second 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: Glass bottles approx. 4 oz size containing 100 mL of 1.5 N KOH.
- Other: The study was terminated on Day 28 by adding 1 mL concentrated HCl to the flasks and shaking was continued for 1 to 3 days. 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 and 28
- Sampling method: For CO2 determination: 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 20mL 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 specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: No
- 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 (%)
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
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
84
% Degr.:
87.2
Parameter:
CO2 evolution
Sampling time:
28 d
Remarks on result:
other: Flask 1 (Normalized to 100% mass balance)
% Degr.:
87.8
Parameter:
CO2 evolution
Sampling time:
28 d
Remarks on result:
other: Flask 2 (Normalized to 100% mass balance)
% Degr.:
87.5
Parameter:
CO2 evolution
Sampling time:
28 d
Remarks on result:
other: Mean of Flask 1,2 (both normalized to 100% mass balance)
Other kinetic parameters:
first order rate constant
Transformation products:
not measured
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
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: 74.3 and 72.3% for Flask 1 and 2 respectively. Because the mass balance in both flasks at the end of the study was less than 90% (85.2 and 82.3% in Flasks 1 and 2 respectively), the results were normalized to 100% mass balance (CO2 in flasks 1 and 2 at end of study was adjusted to 87.2 and 87.8% respectively, normalized basis). For details see ‘Table1’ below in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Not applicable

Biodegradation Test of Alkyl Ethoxy Sulfate (C14E3S) (study # 35451)

Table 1: Flask #1

Day

Indirect %T14C02

Direct %T14C02

Biomass %14C

Solution %14C

Mass Balance

1

10.1

13.0

1.0

88.9

102.9

3

47.5

30.9

13.7

38.8

83.4

5

77.4

55.8

13.9

8.7

78.4

7

83.3

57.3

10.7

6.0

74.0

10

87.3

68.7

8.6

4.2

81.5

14

90.1

66.4

6.7

3.2

76.3

21

90.7

72.9

6.6

2.7

82.2

28

91.1

74.3

6.5

2.4

83.2

28 (normalized to 100% mass balance)

87.2

7.8

2.9

100

*Flask was acidified following completion of sampling.

Table 2: Flask #2

Day

Indirect %T14C02

Direct %T14C02

Biomass %14C

Solution %14C

Mass Balance

1

3.2

13.0

0.9

96.0

109.9

3

41.9

26.9

10.7

47.4

85.0

5

70

54.3

16.0

14.0

84.3

7

78.3

61.9

11.6

7.6

81.1

10

79.2

70.3

14.5

6.3

91.1

14

84.2

75.3

11.2

4.7

91.2

21

84.1

70.2

11.9

4.0

86.1

28

90.6

72.3

6.2

3.1

81.6

28(normalized to 100% mass balance)

87.8

7.6

3.8

100

*Flask was acidified following completion of sampling.

 This is the mass balance calculated by adding the direct T%14CO2, Biomass%14C and solution %14C.

Validity criteria fulfilled:
yes
Conclusions:
Alkyl Ethoxy Sulfate (C14E3S) underwent complete mineralization in surface water. At Day 28, the %CO2 produced was 87.5 (mean of flask 1 and 2). The rate constant for mineralization in surface water was 0.38 day-1 (mean of flask 1 and 2).
Executive summary:

A simulation of the mineralization of Alkyl Ethoxy Sulfate (AES) in surface water was conducted under aerobic conditions in accordance with the OECD 314D guideline. Radiolabeled Alkyl Ethoxy Sulfate (C14E3S) was tested at 100 µg active/L. The river water was collected from Brandywine Creek in Downingtown, Pennsylvania. The test was run in duplicate.  Activated sludge (collected from same location) was also added to each test flask (1 ml sludge/1000 ml river water).

After 28 days, the cumulative percent of theoretical 14CO2 produced was 87.5% (mean of flasks 1 and 2 having values 87.2 and 87.8 respectively.

The rate constant for biodegradation of Alkyl Ethoxy Sulfate (C14E3S) in effluent-surface water was 0.38 day-1(mean of flasks 1 and 2 having values 0.33 and 0.42 day-1respectively).

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: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 Sep 1993 - 19 Jan 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Followed guideline, well documented, with GLP
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314B. Deviations, reliability, and validity evaluated against current OECD 314B (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
according to EPA principles of GLP
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 on Sept. 22, 1993 from Downingtown Regional Water Pollution Control Center, Downingtown, Pennsylvania. This sewage treatment plant receives predominantly domestic waste.
- Storage conditions: Activated sludge was held overnight with aeration.
- Storage length: Overnight
- Preparation of inoculum for exposure: The sludge was screened through approx. 2 mm sieve. The total suspended solids (TSS) concentration of the inoculum was adjusted by settling and decanting the appropriate quantity of supernatant (to concentrate) or by diluting with the settled mixed liquor.
- Concentration of sludge: The sludge was adjusted to a target total suspended solids level of 2500 mg/L
Duration of test (contact time):
28 d
Initial conc.:
1 000 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 1L
- Composition of medium: Each 2 L test flask contained:
1 L activated sludge (2500 mg SS/L)
1000 µg active/L dosing solution (containing mixture of 898 µL of labeled and 1050 µL of unlabeled test material)
- Test temperature: 22 ± 3 °C
- pH: Not reported
- Aeration of dilution water: Yes, please see "Method used to create aerobic conditions" below.
- Suspended solids concentration: 2500 mg/L
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L Erlenmeyer flasks
- Number of culture flasks/concentration: Duplicate
- Method used to create aerobic conditions: The flasks were placed on a rotary platform shaker and aerated continuously with a CO2 free air source. The speed of the shaker was adjusted to keep the sludge solids in suspension but not cause sludge to reach the top of the flasks. Air was purged through the scrubbing train at a constant rate which is adequate to provide 1-2 bubbles/second 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
- Test performed in open system: No
- 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 28 by adding 10 mL concentrated HCl to the flasks and and shaking was continued for 1 to 2 days. Sampling was done as reported in "Sampling method" below with the exception that a new alkali trap was not added and acidification of the fIltrate and incubation in biometer flasks was not required.

SAMPLING
- Sampling frequency: On Day 0.4, 1, 3, 5, 7, 10, 14, 21 and 28
- Sampling method: For CO2 determination: The first alkali trap in the train was removed and a one mL aliquot was counted by LSC in 20 mL of Cab-O-Sil. The two remaining traps were moved one slot closer to the flask and a new trap was added to the third slot. At the same time, base traps are sampled; 10 mL sludge samples were withdrawn and filtered through three 0.45 µm filters. The filter sets were washed with 5 mL IPA/water (50/50) mixture, air dried and counted by LSC in 20mL 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 specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: No
- 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 (%)
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
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
91
% Degr.:
91.4
Parameter:
CO2 evolution
Sampling time:
28 d
Remarks on result:
other: Flask 1 (Normalized to 100% mass balance)
% Degr.:
85.3
Parameter:
CO2 evolution
Sampling time:
28 d
Remarks on result:
other: Flask 2
Other kinetic parameters:
first order rate constant
Transformation products:
not measured
Details on transformation products:
Not determined
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
TEST CONDITIONS
- Aerobicity, 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: 81.8 and 85.3% for Flask 1 and 2 respectively. Because the mass balance in flask 1 at the end of the study was less than 90% (89.5%), the result was normalized to 100% mass balance (%CO2 in flask 1 at end of study was adjusted to 91.4%, normalized basis).
For details see ‘Table1’ below in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION- % of the applied radioactivity present as volatile organics at end of study: Not applicable

Table 1: Biodegradation test of Alcohol ethoxy sulfate (Study # 35452)

Flask 1

Time (Day)

Solution % 14C

Biomass % 14C

Total % 14CO2 per sampling

Mass Balance (%)

0.4

0.7

10.4

46.8

57.9

1

0.8

9.3

59.4

69.5

3

0.4

7.8

65.9

74.1

5

0.5

7.3

70.0

77.8

7

0.3

7.1

72.4

79.8

10

0.3

6.0

74.6

80.9

14

0.4

6.0

76.7

83.1

21

0.7

4.4

78.9

84.0

28

0.7

4.5

81.7

86.9

30

2.1

5.6

81.8

89.5

 Normalized to 100% mass balance

91.4

100

 

  

Flask 2

Time (Day)

Solution % 14C

Biomass % 14C

Total % 14CO2 per sampling

Mass Balance (%)

0.4

0.4

11.7

36.8

48.9

1

0.5

8.6

57.7

66.8

3

0.3

9.2

68.1

77.6

5

0.3

7.7

72.0

80

7

0.3

7.2

74.5

82

10

0.5

6.5

76.9

83.9

14

0.4

5.5

79.1

85

21

1.2

4.6

81.9

87.7

28

0.5

4.3

85.4

90.2

30

2.4

6.1

85.3

93.8

*Flask was acidified following completion of sampling.

This is the mass balance calculated by adding the direct Total %14CO2 per sampling, Biomass %14C and solution %14C

Validity criteria fulfilled:
yes
Conclusions:
Alkyl Ethoxy Sulfate (C14E3S) underwent complete mineralization in activated sludge. At Day 28, the %CO2 produced was 88.3 (mean of flask 1 and 2). The rate constant for mineralization in surface water was 1.75 day-1 (mean of flask 1 and 2).
Executive summary:

A simulation of the mineralization of Alkyl Ethoxylate Sulfate in activated sludge was conducted under aerobic conditions in accordance with the OECD 314B guideline. A solution of radiolabeled and unlabeled Alkyl Ethoxylate Sulfate (C14E3S) was tested at 1000 µg active/L. The activated sludge was collected from Downingtown Regional Water Pollution Control Center, Downingtown, Pennsylvania. This wastewater treatment plant receives predominantly domestic waste. The test treatments were measured in duplicate.

After 28 d, the cumulative percent of theoretical 14CO2 produced was 88.3% (mean of flasks 1 and 2 having values of 91.4 and 85.3 respectively).

The rate constant for biodegradation of Alkyl Ethoxylate Sulfate in activated sludge was 1.75 day-1(mean of flasks 1 and 2 having values of 2.1 and 1.4 day-1respectively).

This biodegradation 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: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 Jul - 01 Aug 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
equivalent or similar to
Guideline:
other: OECD 314C. Deviations, reliability, and validity evaluated against current OECD 314C (Oct. 3, 2008)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA GLP
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Inoculum or test system:
digested sludge
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: Anaerobic digester sludge was collected from Glendale Wastewater Treatment Facility (Cincinnati, OH). This waste water treatment facility receives predominantly domestic waste.
- Storage conditions: The sludge was transferred to anaerobic chamber (Forma Scientific, Model 1024) containing oxygen-free nitrogen.
- Storage length: Not specified
Duration of test (contact time):
120 h
Initial conc.:
1 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CH4 evolution
CO2 evolution
test mat. analysis
other: Biomass ... (see attached file)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 300 mL
- Composition of medium: Each 500 mL glass bottle contained:300 mL sludge mixture (sludge was mixed with an equal volume of mineral salts solution)1 mg/L of test material
- Solubilising agent (type and concentration if used): Not used
- Test temperature: 35°C
- pH: Not reported
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 500 mL glass bottle
- Number of culture flasks/concentration: One for biotic control and one for abiotic control
- Method used to create anaerobic conditions: Not specified
- Measuring equipment: CO2 measuring apparatus was not employed in the study. Mineralization to 14CO2 was determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic treatments.
- Test performed in closed vessels: Yes
- Details of trap for CO2 and volatile organics if used: Alkali traps for absorbing evolved gas (CO2 and CH4) were not used in the study as mineralization was determined by quantifying the production of 14CO2 and 14CH4 by difference relative to the abiotic control.

SAMPLING
- Sampling frequency: Not reported
- Sampling method: At each sampling, each sludge mixture was mixed thoroughly and sampled as follows:
a) For analysis of total radioactivity: 1 mL samples of the mixed sludge were removed and transferred to microcentrifuge tubes acidified, allowed to sit overnight and centrifuged (12,000 g; 15 min).
b) For analysis of parent compound and metabolite: 5 mL samples of the mixed sludge were removed and immediately flash frozen in a dry ice-acetone bath. The frozen sludge samples were lyophilized using a Virtis Benchtop freeze dryer.
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes (sludge sample was autoclaved and amended with mercuric chloride (1g/L) to serve as an abiotic control)
- Toxicity control: No

STATISTICAL METHODS: Statistical analyses included calculation of means. Mineralization data was fitted to a nonlinear kinetic model to determine first-order rate constants.
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
95.3
% Degr.:
> 91
Parameter:
other: 14CO2 and 14CH4 evolution (mineralization)
Sampling time:
21 h
% Degr.:
0
Parameter:
other: % of radioactivity as parent and metabolite
Sampling time:
21 h
% Degr.:
4.3
Parameter:
other: % of radioactivity associated with solids
Sampling time:
21 h
Compartment:
water
DT50:
2.6 h
Type:
other: First order
Remarks on result:
other: Half-life of primary biodegradation
Compartment:
water
DT50:
3 h
Type:
other: First order
Remarks on result:
other: Half-life of ultimate biodegradation
Transformation products:
yes
No.:
#1
Details on transformation products:
In the bioactive sample, the amount of test substance decreased very rapidly with the transient appearance of a metabolite and a concurrent loss of radioactivity (due to mineralization of the test material). The metabolite, chromatographed as alkyl ethoxylate (C14E3), was formed in small amounts in the bioactive samples but rapidly disappeared. No other metabolites were observed at any time.
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Details on results:
TEST CONDITIONS
- Anaerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS: No major transformation products were produced during the mineralization.

MINOR TRANSFORMATION PRODUCTS: Alkyl ethoxylate (C14E3) was formed in small amounts in the bioactive samples but rapidly disappeared. No other metabolites were observed at any time. Transient appearance of Alkyl ethoxylate concurrent with the initial disappearance of C14E3S indicated that the initial step in the biodegradation process was sulfatase mediated hydrolysis of the sulfate group.

EXTRACTABLE RESIDUES: In the abiotic control, nearly all (92-100%) of the radioactivity extracted into methanol and chromatographed as parent Alkyl ethoxylate sulfate.
- % of applied amount at Day 0: Approx. 90% in bioactive flask (parent; sample time was 0 h)
- % of applied amount at end of study period: 0% in bioactive flask (parent; sample time was 120 h)

NON-EXTRACTABLE RESIDUES: A very small fraction (<10%) was nonextractable in the abiotic control
- % of applied amount at Day 0: 0 % in bioactive flask (sample time was 0 h)
- % of applied amount at end of study period: approx. 10% in bioactive flask (sample time was 120 h)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: >91% in bioactive test flask (sample time was 120 h). CO2 evolution was not analyzed for abiotic flask as radioactivity in the abiotic control was recovered primarily as intact parent.

VOLATILIZATION: No volatilization of test material was observed as the TLC analysis of the abiotic control revealed that the parent C14E3S was recovered intact.
Validity criteria fulfilled:
yes
Conclusions:
C14E3S (Alkylethoxysulfate, AES) underwent mineralization in anaerobic digester sludge with half-lives less than a few hours. After 21 h, greater than 91% was mineralized, 4.3% was non-extractable (solids), and 0% remained as either parent compound or metabolite. The half -lives for primary biodegradation and ultimate biodegradation were 2.6 and 3 h respectively.
Executive summary:

A simulation of the mineralization of Alkyl ethoxy sulfate (C14E3S) in anaerobic digester sludge was conducted in accordance with OECD 314C guideline. A solution of radiolabeled and non-radiolabeled alkyl ethoxy sulfate was tested at 1 mg/L. The inoculum was anaerobic digester sludge collected from Glendale Wastewater Treatment Facility. This treatment facility receives predominantly domestic waste.

After 21 h, greater than 91% was mineralized, 4.3% was non-extractable (solids), and 0% remained as either parent compound or metabolite. The transient appearance of alkyl ethoxylate (C14E3) concurrent with the initial disappearance of parent Alkyl ethoxy sulfate C14E3S indicated that the initial step in the anaerobic biodegradation of this compound was sulfatase mediated hydrolysis of the sulfate group.

The half- lives for biodegradation of alkyl ethoxy sulfate in anaerobic digester sludge were:

Half-life for primary biodegradation: 2.6 h

Half life for ultimate biodegradation: 3 h

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

Description of key information

According to the REACH regulation ANNEX IX column 2, biodegradation simulation studies needs not to be conducted if the test item is readily biodegradable. Furthermore, biodegradation simulation studies are not a data requirement for the current tonnage band.

Key value for chemical safety assessment

Additional information

According to the REACH regulation ANNEX IX column 2, studies on the biodegradation in water and sediment need not be conducted if the test item is readily biodegradable.

However, data are available for the fate of alcohol ethoxysulfates and are presented below.

In a test conducted to OECD 314 C the alcohol ethoxysulfate C14E3S was incubated in anaerobic digester sludge. 91% of the radiolabelled test substance was mineralized after 21 hours. Alkylethoxylate C14EO3 occurred together with the disappearance of the parent compound. Therefore it is proposed that the initial step in anaerobic degradation is sulfatase mediated hydrolysis of the sulfate group. The half-life for primary degradation was determined as 2.6 hours (P&G, 1994b). This result was supported by a similar test with alcohol ethoxysulfate C14E3S according to OECD 314 C. After 14 days 88% of the applied radioactivity was recovered in evolving gases. The half-life was determined to be 0.67 d (P&G, 1994b).

In two die-away studies according to OECD guideline 314 A and 314 B (P&G, 1998) alcohol ethoxysulfates were removed from the exposure matrix to an extent of 95.2% and 88.4% after 24 hours (mineralization and incorporation into solids). The degradation rates determined for mineralization were 0.033 min-1 and 0.003 min-1, respectively. 98.8% of the test substance was removed from raw sewage after 24 hours. 65.7% were converted to a metabolite and 33.1% were incorporated into solids.

Another study (1994, P&G) investigated the degradation of alcohol ethoxysulfates according to OECD 314 B. In activated sludge treating predominately domestic sewage under aerobic conditions radiolabeled C14E3S was mineralized to 88.3% after 28 days of exposure. Therefore, the rate constant for biodegradation in activated sludge was 1.75 d-1.

In surface water (89% river water, 10% sewage treatment plant effluent and 1% activated sludge) 39.7% of alcohol ethoxysulfates were mineralized after 48 hours. 60.3% were non extractable residues (P&G, 1996). Degradation rates could be calculated as 0.97 h-1 for primary degradation, 0.061 h-1 for ultimate biodegradation and 0.028 h-1 for mineralization.

In another simulation test in surface water according to OECD guideline 314 D 87.5% of theoretical radiolabeled 14CO2 were observed after 28 days of exposure. The degradation rate constant derived for biodegradation of C14E3S was 0.38 d-1 (P&G, 1994).

Taking into account all available results obtained from the different simulation tests conducted according to OECD guideline 314 it was demonstrated, that alcohol ethoxysulfates are not persistent in water and are ultimately removed under aerobic and anaerobic conditions.