Amines, C14-16 (even numbered)-alkyldimethyl, N-oxides

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 21, 1995 to March. 28, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 314C. Deviations, reliability, and validity evaluated against current OECD 314C (Oct. 3, 2008)

Deviations:

no

GLP compliance:

yes

Remarks:

USEPA GLP (40 CFR Part 792)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

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: The acclimated sludge was harvested from a bench scale anaerobic acclimation reactor. The sludge also contained waste sludge from the acclimation reactor and unacclimated anaerobic feed sludge containing activated sludge solids as a substrate.
- Storage conditions: The sludge was maintained in the anaerobic acclimation reactor for 15 weeks before employing it in the test.
- Pretreatment: 100 mL sludge was removed daily from anaerobic acclimation reactor and replaced by 90 mL of feed mixture and 10 mL of test substance solution (prepared in deionized water). The initial concentration of test substance was 1 mg active/L. The concentration of test substance in stock solution was 10 mg active/L for the first week and then was constant at 50 mg active/L for the rest of the acclimation period.
- Concentration of sludge: 300 mL of sludge solution (50% digester sludge and 50% mineral salts solution)
- Initial cell/biomass concentration: Not reported
- Water filtered: No
- More details on the source of sludge, operation of reactor, and preparation of inoculum can be found in the “any other information on materials and methods’ section.

Duration of test (contact time):

7 d

Initial conc.:

1 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CH4 evolution

CO2 evolution

test mat. analysis

other: (metabolite)

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 314C guideline.
- Solubilising agent: Not used
- Test temperature: The test bottles were incubated at 35 °C anaerobic chamber throughout the test period.
- pH: Not reported
- Aeration of dilution water: No. The test was conducted under anaerobic conditions.
- Suspended solids concentration: 20,730 mg TS/L
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 500 mL glass media bottles containing 300 mL test sludge mixture (50% digester sludge and 50% mineral salts solution) and 2500 µL dosing solution.
- Number of culture flasks/concentration: Two test bottles per concentration
- Method used to create anaerobic conditions: The test bottles were placed inside the anaerobic chamber.
- 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 open system: No
- 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: At 1-5, 15, 30, 60, and 90 minutes, 3,4.5, and 6 h of Day 1 and on 2, 3,4,5, 6 and 7d.
- Sampling method: At each sampling, each sludge mixture was mixed thoroughly and sampled as follows:
a) For determination of evolved CO2 and CH4: Triplicate 1 mL samples of the mixed sludge were removed and transferred to microcentrifuge tubes containing 25 µL of conc. HCI, vortexed and allowed to sit overnight or longer.
b) For analysis of parent compound and radioactivity associated with solids: A single 5 mL sample of the mixed sludge was removed transferred to screw-top glass culture tubes and immediately flash frozen in a dry ice/acetone bath. The frozen sludge samples were stored at -80°C until lyophilization.
Analysis of sample is discussed in ‘Details on analytical method’ section.
- Sterility check if applicable: Not applicable
- 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: For kinetic analysis, the total gas production data were fitted to various equations using Jandel TableCurve 2D software. The equation that provided the best statistical fit was the "three half order without growth" model. The criteria used to judge the best kinetic model for the observed data were: a). r2 b). Examination of error residuals for the regression model. c) Using the highest F-value for the fewest parameters used in a model. When several models have similar r2 values, it is the last two criteria that take precedence in deciding the best fit model.

Reference substance:

not required

Test performance:

No data

Compartment:

other: water, material (mass) balance

% Recovery:

117

St. dev.:

12

% Degr.:

60

Parameter:

other: 14CO2 and 14CH4 evolution (mineralization)

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance

% Degr.:

35.2

Parameter:

other: % of radioactivity as parent

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance

% Degr.:

4.9

Parameter:

other: % of radioactivity associated with solids

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance

Compartment:

other: Anaerobic sludge

DT50:

47.4 h

Type:

other: 3/2 order model without growth

Remarks on result:

other: This is the predicted time required for primary degradation

Compartment:

other: Anaerobic sludge

DT50:

37.8 h

Type:

other: 3/2 order model without growth

Remarks on result:

other: This is the predicted time required for mineralization

Other kinetic parameters:

other: 3/2 order rate constant without growth (provided the best statistical fit) 20.7 + 18.0 h-1 for primary degradation... (see attached file)

Transformation products:

not measured

Details on transformation products:

Not applicable

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

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

EXTRACTABLE RESIDUES: An average 68.7% of radioactivity was extracted and chromatographed as a single peak at Rf 0.36-0.37 in the abiotic control (which corresponded to the parent amine oxide).
- % of applied amount at Day 0: 86.8% in active flask (parent; sample time was 1-5 min)
- % of applied amount at end of study period: Not analyzed at day 7. On day 5, 6.0% of radioactivity was parent compound.

NON-EXTRACTABLE RESIDUES: An average 23.3% of radioactivity was non-extractable and remained with the solids in the abiotic control.
- % of applied amount at Day 0: 8% in active flask (sample time was 1-5 min)
- % of applied amount at end of study period: 5.3% in active flask (day 7)

MINERALISATION
- % of applied radioactivity present as radioactive gases at end of study: 89.9 and 9.7% in active and abiotic test flask respectively. For details, please refer to ‘table 1’ in the ‘Any other information on results incl. tables’ section.

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

OTHERS:
The rate constants for primary biodegradation and mineralization were calculated by various models (such as 3/2 Order without growth, 3/2 Order with growth, First order, first order logistic and first order with lag). The details can be found in the study report.

ABIOTIC CONTROL:
Parameter @79 hours Average (0-7 d)
% evolved as gas 12.4 9.7
% recovered as parent 69.4 68.7
% non-extractable 22.6 23.3
Mass Balance 104.5 103.7

Results with reference substance:

Not applicable

Mineralization test of Amine oxide (study # 36088)

Table 1: Active flask

Sample Time	% Parent	%Gases	% Non extractable	% Mass Balance
1-5 min.	86.8	18.6	8	113.4
15 min.	85	21.1	7.5	113.6
30 min.	83.9	26.4	6.7	117
60 min.	92.3	30.8	22.4	145.5
90 min	76.5	27.5	16.6	120.6
3 hours	76.3	32.6	20.1	129
4.5 hours	74.8	38.2	13.5	126.5
6 hours	72.3	38.4	14.9	125.6
1 Day	55.7	48	9.6	113.3
2 Days	43	62.1	7.6	112.7
3 Days	44.1	75.2	6.1	125.4
4 Days	21.9	78.3	4.3	104.5
5 Days	6	85.1	5.3	101.4
6 Days		88.6	4.7	103.2
7 Days		89.9	5.3	105.8
Mean				117+12
Abiotic Control (mean ± s.d.)	68.7 ± 4.4	9.7 ± 3.8	23.3 ± 1.9	103.7 ± 3.4

Test Substances: E5138.01 and E5119.01

Note: The Days 6 and 7 samples were not analyzed by Rad-TLC due to minimal activity in the bioactive samples at Day 5.

Table 2: Abiotic flask

Sample Time (hours)	% Parent	%Gases	% Non extractable	% Mass Balance
6	70.2	12.3	24.8	107.3
24	63	9.4	25.7	98.1
48	63.7	12.6	NA	NA
79	69.4	12.4	22.6	104.5

NA = Not analysed

The results at 79 h with the biotic flask were normalized to 100% mass balance as follows:

	79 h
Parameter	Measured	Normalized
Gas (CO2 + CH4)	75.2	60.0
Parent	44.1	35.2
Non-extractable	6.1	4.9
Mass Balance	125.4	100

Validity criteria fulfilled:

yes

Conclusions:

Amine oxide underwent mineralization in anaerobic digester sludge. After 3 days, 60% was mineralized, 5% was non-extractable (solids), and 35% remained as parent compound. The rate constants for primary biodegradation and mineralization in anaerobic digester sludge were 20.7 and 3.84 h-1, respectively.

Executive summary:

A simulation of the biodegradation of C12 alkyl amine oxide in anaerobic digester sludge was conducted in accordance with the OECD 314C guideline. A solution of radiolabeled and non-radiolabeled amine oxide was tested at 1 mg/L. The inoculum was acclimated anaerobic digester sludge harvested from a bench scale anaerobic acclimation reactor. The test treatments were measured in duplicate.

After 3 days, 60% of the amine oxide was mineralized, 5% was non-extracable (solids), and 35% remained as parent compound. The rate constants for biodegradation of amine oxide in anaerobic digester sludge were:

    Primary biodegradation: 20.7 h-1

    Mineralization: 3.84 h-1.

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

Reference 2

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 21, 1995 to March. 28, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 314C. Deviations, reliability, and validity evaluated against current OECD 314C (Oct. 3, 2008)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

USEPA GLP (40 CFR Part 792)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

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: The acclimated sludge was harvested from a bench scale anaerobic acclimation reactor. The sludge also contained waste sludge from the acclimation reactor and unacclimated anaerobic feed sludge containing activated sludge solids as a substrate.
- Storage conditions: The sludge was maintained in the anaerobic acclimation reactor for 15 weeks before employing it in the test.
- Pretreatment: 100 mL sludge was removed daily from anaerobic acclimation reactor and replaced by 90 mL of feed mixture and 10 mL of test substance solution (prepared in deionized water). The initial concentration of test substance was 1 mg active/L. The concentration of test substance in stock solution was 10 mg active/L for the first week and then was constant at 50 mg active/L for the rest of the acclimation period.
- Concentration of sludge: 300 mL of sludge solution (50% digester sludge and 50% mineral salts solution)
- Initial cell/biomass concentration: Not reported
- Water filtered: No
- More details on the source of sludge, operation of reactor, and preparation of inoculum can be found in the “any other information on materials and methods’ section.

Duration of test (contact time):

7 d

Initial conc.:

1 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CH4 evolution

CO2 evolution

test mat. analysis

other: (metabolite)

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 314C guideline.
- Additional substrate: None
- Solubilising agent: Not used
- Test temperature: The test bottles were incubated at 35 °C anaerobic chamber throughout the test period.
- pH: Not reported
- Aeration of dilution water: No. The test was conducted under anaerobic chamber.
- Suspended solids concentration: 20,730 mg TS/L
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 500 mL glass media bottles containing 300 mL test sludge mixture (50% digester sludge and 50% mineral salts solution) and 2500 µL dosing solution.
- Number of culture flasks/concentration: Two test bottles per concentration
- Method used to create anaerobic conditions: The test bottles were placed inside the anaerobic chamber.
- 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 open system: No
- 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: At 1-5, 15, 30, 60, and 90 minutes, 3,4.5, and 6 h of Day 1 and on 2, 3,4,5,
6 and 7d.
- Sampling method: At each sampling, each sludge mixture was mixed thoroughly and sampled as follows:
a) For determination of evolved CO2 and CH4: Triplicate 1 mL samples of the mixed sludge were removed and transferred to microcentrifuge tubes containing 25 µL of conc. HCI, vortexed and allowed to sit overnight or longer.
b) For analysis of parent compound and radioactivity associated with solids: A single 5 mL sample of the mixed sludge was removed transferred to screw-top glass culture tubes and immediately flash frozen in a dry ice/acetone bath. The frozen sludge samples were stored at -80°C until lyophilization.
Analysis of sample is discussed in ‘Details on analytical method’ section.
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not reported

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: For kinetic analysis, the total gas production data were fitted to various equations using Jandel TableCurve 2D software. The equation that provided the best statistical fit was the "three half order without growth" model. The criteria used to judge the best kinetic model for the observed data were: a). r2 b). Examination of error residuals for the regression model. c) Using the highest F-value for the fewest parameters used in a model. When several models have similar r2 values, it is the last two criteria that take precedence in deciding the best fit model.

Reference substance:

not required

Test performance:

No data

Compartment:

other: water, material (mass) balance

% Recovery:

110.8

St. dev.:

5.6

% Degr.:

43

Parameter:

other: 14CO2 and 14CH4 evolution (mineralization)

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance.

% Degr.:

53

Parameter:

other: % of radioactivity as parent

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance.

% Degr.:

4

Parameter:

other: % of radioactivity associated with solids

Sampling time:

79 h

Remarks on result:

other: Normalized to 100% mass balance.

Compartment:

other: Anaerobic sludge

DT50:

73.8 h

Type:

other: 3/2 order model without growth

Remarks on result:

other: This is the predicted time required for primary degradation

Compartment:

other: Anaerobic sludge

DT50:

72.7 h

Type:

other: 3/2 order model without growth

Remarks on result:

other: This is the predicted time required for mineralization

Other kinetic parameters:

other: 3/2 order without growth (Provided the best statistical fit) 22.1 ± 32.2 h-1 for primary biodegradation.... (see attached file)

Transformation products:

not measured

Details on transformation products:

Not applicable

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

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

EXTRACTABLE RESIDUES: An average 97.9% of radioactivity was extracted and chromatographed as a single peak at Rf 0.31-0.34 in the abiotic control (which corresponded to the parent amine oxide).
- % of applied amount at Day 0: 93.5% in active flask (parent; sample time was 1-5 min)
- % of applied amount at end of study period: 5.9 % in active flask (parent; sample time was 7 d)

NON-EXTRACTABLE RESIDUES: An average 2.2% of radioactivity was non-extractable and remained with the solids in the abiotic control.
- % of applied amount at Day 0: 4.9% in active flask (sample time was 1-5 min)
- % of applied amount at end of study period: 7.3% in active flask (Day 7)

MINERALISATION
- % of applied radioactivity present as radioactive gases at end of study: 87.5% and 14.1% in active and abiotic test flask respectively. For details, please refer to ‘table 1’ in the ‘Any other information on results incl. tables’ section.

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

OTHERS:
The rate constants for primary biodegradation and mineralization were calculated by various models (such as 3/2 Order without growth, 3/2 Order with growth, First order, first order logistic and first order with lag). The details can be found in the study report.

ABIOTIC CONTROL
Parameter @79 h Average (0-7 d)
% evolved as gas 14.3 14.1
% recovered as parent 92.7 97.1
% non-extractable 2.1 2.2
Mass Balance 109.2 114.3

Results with reference substance:

Not applicable

Mineralization test of Amine oxide (study # 36088)

Table 1: Active flask

Sample Time	% Parent	%Gases	% Non extractable	% Mass Balance
1-5 min.	93.5	20.1	4.9	118.5
15 min.	85.8	17.1	13.7	116.5
30 min.	93.8	21.5	3.3	118.7
60 min.	84.0	19.9	2.8	106.6
90 min	86.3	19.4	2.8	108.5
3 hours	87.3	20.3	1.7	109.3
4.5 hours	90.2	22.1	2.5	114.8
6 hours	86.8	23.8	2.2	112.7
1 Day	84.2	26.3	2.5	113.0
2 Days	69.7	37.4	2.8	110.0
3 Days	61.7	50.0	4.7	116.4
4 Days	37.5	59.7	5.1	102.2
5 Days	15.1	75.7	6.2	106.2
6 Days	8.3	83.0	4.2	101.4
7 Days	5.9	87.5	7.3	106.7
Mean				110.76±5.6
Abiotic Control (mean ± s.d.)	97.1 ± 3.3	14.1 ± 3.1	2.2 ±0.6	114.3 +4.3

Test substances: E5138.01 and E5139.01

Table 2: Abiotic flask

Sample Time (hours)	% Parent	%Gases	% Non extractable	% Mass Balance
6	NA	17.1	3.2	114.1
24	96.4	12.7	2.2	111.3
48	95.1	15.8	NA	NA
79	92.7	14.3	2.1	109.2

The results at 79 h with the biotic flask were normalized to 100% mass balance as follows:

	79 h
Parameter	Measured	Normalized
Gas (CO2 + CH4)	50.0	43
Parent	61.7	53
Non-extractable	4.7	4
Mass Balance	116.4	100

Validity criteria fulfilled:

yes

Conclusions:

Amine oxide underwent mineralization in anaerobic digester sludge. After 3 d, 43% was mineralized, 4% was non-extractable (solids), and 53% remained as parent compound. The rate constants for primary biodegradation and mineralization in anaerobic digester sludge were 22.1 and 79.5 h-1 respectively.

Executive summary:

A simulation of the biodegradation of C12 amine oxide in anaerobic digester sludge was conducted in accordance with OECD 314C guideline. A solution of radiolabeled and non-radiolabeled amine oxide was tested at 1 mg/L. The inoculum was acclimated anaerobic digester sludge harvested from a bench scale anaerobic acclimation reactor. The test treatments were measured in duplicate.

After 3 d, 43% was mineralized, 4% was non-extractable (solids), and 53% remained as parent compound. The rate constants for biodegradation of amine oxide in anaerobic digester sludge were:

Primary biodegradation: 22.1 h-1

Mineralization: 79.5 h-1

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

Reference 3

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 21, 1995 to Feb. 22, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 314C. Deviations, reliability, and validity evaluated against current OECD 314C (Oct. 3, 2008)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

USEPA GLP (40 CFR Part 792

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

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: The acclimated sludge was harvested from a bench scale anaerobic acclimation reactor. The sludge also contained waste sludge from the acclimation reactor and unacclimated anaerobic feed sludge containing activated sludge solids as a substrate.
- Storage conditions: The sludge was maintained in the anaerobic acclimation reactor for 8 weeks before employing it in the test.
- Pretreatment: 100 mL sludge was removed daily from anaerobic acclimation reactor and replaced by 90 mL of feed mixture and 10 mL of test substance solution (prepared in deionized water). The initial concentration of test substance was 1 mg active/L. The concentration of test substance in stock solution was 10 mg active/L for the first week and then was constant at 50 mg active/L for the rest of the acclimation period.
- Concentration of sludge: 300 mL of sludge solution (50% digester sludge and 50% mineral salts solution)
- Initial cell/biomass concentration: Not reported
- Water filtered: No
- More details on the source of sludge, operation of reactor, and preparation of inoculum can be found in the “any other information on materials and methods’ section.

Duration of test (contact time):

62 d

Initial conc.:

1 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CH4 evolution

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 314C guideline.
- Solubilising agent: Not used
- Test temperature: The test bottles were incubated at 35±2 °C in a water bath throughout the test period.
- pH: Not reported
- Aeration of dilution water: The test was conducted under anaerobic condition. The headspace of the test bottles was purged continuously with nitrogen at a rate of approx. 5 mL/min.
- Suspended solids concentration: 31,013 mg TS/L
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 500 mL glass media bottles containing 300 mL test sludge mixture (50% digester sludge and 50% mineral salts solution) and 100 µL dosing solution.
- Number of culture flasks/concentration: 2 test bottles per concentration
- Method used to create anaerobic conditions: The test bottles were placed inside the anaerobic chamber and 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 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 open system: No
- Details of trap for CO2 and volatile organics if used: French square 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: The test systems were acidified with 10 mL 5N H2SO4 by injected through a sampling port on Day 61. One final set of base trap samples was collected and analyzed next day. The N2 purging of the headspace of the bottles continued overnight.

SAMPLING
- Sampling frequency: At Day 1, 2, 3, 5, 7, 10, 14, 21, 28, 35, 43, 50, 57, 61 and 62
- 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.
- Sterility check if applicable: N ot applicable
- Sample storage before analysis: Not specified

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

STATISTICAL METHODS: The total gas production data were fitted to various equations for kinetic analysis using Jandel TableCurve 2D software. The equation that provided the best statistical fit was the "three half order without growth" model. The criteria used to judge the best kinetic model for the observed data were: a). r2 b). Examination of error residuals for the regression model. C) Using the highest F-value for the fewest parameters used in a model.

Reference substance:

not required

Test performance:

No data

Compartment:

other: water, material (mass) balance

% Recovery:

103.7

St. dev.:

1.2

% Degr.:

76

Parameter:

other: 14CO2 and 14CH4 evolution

Sampling time:

62 d

Remarks on result:

other: In test replicate 1 (The evolved gases were recovered as 14C-gas)

% Degr.:

74.9

Parameter:

other: 14CO2 and 14CH4 evolution

Sampling time:

62 d

Remarks on result:

other: In test replicate 2 (The evolved gases were recovered as 14C-gas)

Compartment:

other: Anaerobic sludge

DT50:

14.3 d

Type:

other: 3/2 order model with growth

Remarks on result:

other: Half-life of mineralization

Other kinetic parameters:

other: 3/2 order with growth (Provided the best statistical fit ) 0.055±0.005 h-1 for mineralization

Transformation products:

not measured

Details on transformation products:

Not applicable

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

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

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 radioactive gases at end of study: 76 and 74.9% in test replicate 1 and 2 respectively.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics (CH4) at end of study: determined after conversion to CO2

OTHERS:
The rate constants were calculated by various models such as 3/2 Order without growth, 3/2 Order with growth, First order, first order logistic and first order with lag. The details can be found in the study report.

Results with reference substance:

Not applicable

Table 1: Mineralization test of Amine oxide (study # 36088)

	Cumulative % of dosed 14C (mean ± standard deviation)
Sample time (Day)	14CO2	14CH4	Total
1	2.1 ± 0.1	1.5±0.1	3.6 ± 0.2
2	4.0 ± 0.1	2.6 ± 0.1	6.5 ± 0.1
3	6.7 ± 0.1	3.7 ± 0.0	10.4 ± 0.1
5	12.1 ± 0.6	6.4 ± 0.1	18.5 ± 0.5
7	20.8 ± 0.0	9.0 ± 0.4	29.8 ± 0.4
10	28.6 ± 0.3	10.4 ± 0.8	39.0 ± 0.6
14	36.3 ± 0.4	12.1 ± 1.0	48.4 ± 0.6
21	44.3 ± 1.3	14.3 ± 0.8	58.6 ± 0.4
28	48.5 ± 0.8	15.3 ± 0.8	63.8 ± 0.1
35	51.4 ± 0.9	15.9 ± 0.8	67.2 ± 0.1
43	53.4 ± 0.9	16.3 ±0.7	69.7 ± 0.2
50	54.6 ± 0.8	16.7 ± 0.6	71.3 ± 0.3
57	55.6 ± 0.8	17.0 ±0.4	72.6 ± 0.4
61	56.2 ± 0.8	17.2 ± 0.4	73.4 + 0.4
62	57.7±0.5	17.3 ± 0.3	75.0 ± 0.2

Test compounds: E5138.01 and E5139.01

Validity criteria fulfilled:

yes

Conclusions:

Amine oxide underwent mineralization in anaerobic digester sludge. At Day 62, 75.0% of the amine oxide was either CO2 or CH4. The rate constant for mineralization in anaerobic digester sludge was 0.055 h-1.

Executive summary:

A simulation of the mineralization of C12 alkyl amine oxide in anaerobic digester sludge was conducted in accordance with OECD 314C guideline. A solution of radiolabeled and non-radiolabeled amine oxide was tested at 1 mg/L. The radiolabel (14C) was on the methylene groups (CH2) of the alkyl side-chain.

The inoculum was acclimated anaerobic digester sludge harvested from a bench scale anaerobic acclimation reactor. The test treatments were measured in duplicate. The evolution of both 14C-CO2 and 14C-methane was measured.

The mean cumulative percent of theoretical % 14C-gas produced was 75.5.

The rate constant for mineralization of amine oxide in anaerobic digester sludge was 0.055 h^-1

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

Reference 4

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-11-7 to 1995-11-21 (14C-DDAO Die-away Study); 1995-08-17 to 1995-10-18 for the related CAS Study from which the effluent mixture was taken.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Not to GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 314 D (Simulation Tests to Assess the Biodegradability of Chemicals in Wastewater. D: Biodegradation in Treated Effluent-Surface Water Mixing Zone)

Deviations:

no

Principles of method if other than guideline:

Study predates but is similar to Guideline 314D. This study was preceded by a Continuous Activated Sludge test (See Cross Reference to Other Study), and used effluent from the CAS study to run this effluent die-away study. The goal of this test was to determine the rate and extent of biodegradation of the remaining test substance and its metabolites following activated sludge wastewater treatment.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
N/A

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: natural water/effluent from CAS test/activated sludge, non-adapted

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): River water was collected from the Zenne River sampled at Tubize.
- Storage conditions: Refrigerated at 4 deg C until use.
- Storage length: N/A
- Temperature (°C) at time of collection: N/A
- pH at time of collection: N/A; mixture of effluent, river water and activated sludge, 7.8
- Electrical conductivity: N/A
- Redox potential (mv) initial/final: N/A
- Oxygen concentration (mg/l) initial/final: N/A
- Hardness (CaCO3): N/A
- Dissolved organic carbon (%): N/A
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): N/A
- Water filtered: N/A
- Type and size of filter used, if any: N/A

Effluent - The source of sewage effluent were 2 continuous activated sludge (CAS) units that were used to evaluate the biodegradation of the test substance. Two CAS units, fed either 110 ug/L or 384 ug/L of test substance, using raw sewage (mostly domestic) collected from Bocholt, Belgium, enriched with synthetic sewage, and run for 21-27 days. See companion study #35936 and its IUCLID summary.

Details on source and properties of sediment:

N/A

Details on inoculum:

- Source of inoculum/activated sludge: from the aerators of a treatment plant in Hofstade dealing primarily with domestic sewage
- Laboratory culture: no data
- Method of cultivation: no data
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: no data
- Pretreatment: no data
- Concentration of sludge: no data
- Initial cell/biomass concentration: no data

Duration of test (contact time):

14 d

Initial conc.:

2 µg/L

Based on:

other: effluent CAS unit 1

Initial conc.:

1 µg/L

Based on:

other: effluent CAS unit 2

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 10 L
- Composition of medium: 3.3 L of effluent mixture from each of the two CAS test units was diluted with 6.6 L river water and 100 mL of activated sludge in a 10 L test container. Abiotic controls were prepared in the same way and 0.1 g/L HgCl2 was added to these test containers to kill microbial activity. The composition of the river water die away test was as follows:
6.6 L river water
3.3 L effluent (from CAS unit 1 or 2)
0.1 L activated sludge (from a STP in Hofstade)
10.0 L Total

- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 20-21 deg C
- pH: 7.8 mixture of effluent, river water and activated sludge.
- pH adjusted: no
- CEC (meq/100 g): N/A
- Aeration of dilution water: N/A
- Suspended solids concentration: N/A
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: N/A
- Other: N/A


TEST SYSTEM
- Culturing apparatus: 10 L test containers, covered with a watchglass and placed on a rotary platform shaker. Gently mixed during the entire test period.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: gentle mixing via platform shaker.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: N/A


- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: N/A
- Other: pH and dissolved oxygen readings were taken daily during the test.


SAMPLING
- Sampling frequency: Samples were taken at the start and after 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 24, 48, 72, 120, 168, 240, and 336 hours.
- Sampling method: Test vessels were set on a magnetic stir plate and stirred prior to sampling.
- Sterility check if applicable: N/A
- Sample storage before analysis: LSC triplicate samples (10 mL) were acidified. TLC-RAD samples were flash frozen immediately after sampling.
- Other: N/A


CONTROL AND BLANK SYSTEM
- Inoculum blank: N/A
- Abiotic sterile control: Abiotic controls were sampled and analyzed the same as the test samples.
- Toxicity control: N/A
- Other: N/A


STATISTICAL METHODS: See 'Remarks on results including tables and figures' below.

Reference substance:

not required

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: Average mass balance of unit 1 = 87% (+/- 21%); unit 2 = 99% (+/- 17%).

% Degr.:

43

Parameter:

CO2 evolution

Sampling time:

14 d

Remarks on result:

other: Unit 1

% Degr.:

39

Parameter:

other: % of radioactivity as parent

Sampling time:

14 d

Remarks on result:

other: Unit 1

% Degr.:

9

Parameter:

other: % of radioactivity associated with solids

Sampling time:

14 d

Remarks on result:

other: Unit 1

% Degr.:

63

Parameter:

CO2 evolution

Sampling time:

14 d

Remarks on result:

other: Unit 2

% Degr.:

24

Parameter:

other: % of radioactivity as parent

Sampling time:

14 d

Remarks on result:

other: Unit 2

% Degr.:

24

Parameter:

other: % of radioactivity associated with solids

Sampling time:

14 d

Remarks on result:

other: Unit 2

Compartment:

entire system

DT50:

4.3 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Half-life of mineralization (Unit 1)

Compartment:

entire system

DT50:

2.1 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Half-life of mineralization (Unit 2)

Transformation products:

not specified

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not specified

Details on results:

See Remarks below

Results with reference substance:

N/A

Results

The mixture of effluent, river water and activated sludge was analyzed by LSC and TLC-RAD to determine the speciation in the starting solution:

	Int. 1 (ug/L)	Int. 2 (ug/L)	Int. 3 (ug/L)	Int. 4 (ug/L)	Parent (ug/L)	Int. 5 (ug/L)
Test Unit 1	<0.33	<0.33	<0.33	<0.33	2	<0.33
Test Unit 2	<0.33	<0.33	2.5	0.4	1	<0.33

 

The CO2 production was calculated as follows:

 

% mineralized=(1-dpm/mL in acidified bioactive samples) x 100

                                  dpm/mL in acidified abiotic control

                                               

The results of the CO2 production indicate that a plateau was reached corresponding to a CO2 production of 43% in Test Unit 1 and 63% in Test Unit 2.

The percent 14CO2 production versus time is calculated as follows:

                               14CO2= a (1-e^-k(t-c))

                                               Where a =asymptote of curve (%TCO2)

                                                               k= first order rate constant (day-1)

                                                               t= time of incubation (days)

                                                               c= lag period (if any) before CO2 production occurs (days)

                                                               

Test Unit	Final % TCO2	Asymptote (a) % TCO2	Rate k	Lag c	T 1/2
1	43	46	0.16 day-1	0 hours	4.3 days
2	63	63	0.33 day-1	0.6 hours	2.1 days

 

  Average mass balances over the test period

Test Unit	% mass balance	% rstd
1	87	21
2	99	17

Validity criteria fulfilled:

yes

Conclusions:

After simulated activated sludge treatment, amine oxide continued to biodegrade in a river water die away study. After 14 days, 53% was mineralized, 32% was parent, and 16% was associated with solids (average of 2 tests). The half-life of mineralization was 4.3 days (in test unit 1) and 2.1 days (in test unit 2).

Executive summary:

The biodegradation of dodecyl amine oxide (C12 AO) was evaluated in a River-Water Die Away study according to the OECD 314D guidelines. The test system consisted of river water, effluent from 2 CAS units, and a small amount of activated sludge from a treatment plant handling primarily domestic sewage. The test period was 14 days, and 14CO2 production and radiochemistry were measured. The river water die away (RDA) test was done in duplicate: RDA test 1 used effluent from CAS unit 1, and RDA test 2 used effluent from CAS unit 2. RDA test 1 was dosed with 2 ug/L test substance (from the effluent from CAS unit 1). RDA test 2 was dosed with 1 ug/L test substance, and 2.9 ug/L metabolites of parent (from the effluent from CAS unit 2).

After 14 days, the total 14CO2 production in the river die away study was 43% for RDA test 1, and 63% for test unit 2. The half-life of mineralization was 4.3 days in RDA 1, and 2.1 days in RDA 2.

After 14 days, loss of parent was 61% in RDA test 1. The % loss of parent in unit 2 was unreliable (7%), because of the presence of significant metabolites at the start in RDA test 2. After 14 days, the amount of parent compound was 0.78 ug/L in RDA test 1, and 0.92 ug/L in RDA test 2. Intermediates were mineralized to below detection limits of the TLC-RAD method (<0.33 ug/L).

The radiochemical mass balances averaged 87 and 99% for RDA tests 1 and 2 respectively. After 14 days in RDA test 1, 43% of the radioactivity was CO2, 39% was parent, and 9% was associated with the solids (total=91%). After 14 days in RDA test 2, 63% of the radioactivity was CO2, 24% was parent, and 24% was associated with the solids (total=111%).

This biodegradation study satisfies the guideline requirements for the OECD 314D river water die away test.

Reference 5

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995-08-17 to 1995-10-18 (DDAO CAS Study), and 1995-11-6 to 1995-12-6 (14C-DDAO Dieaway Study)

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

In prinicple, the study followed the OECD 303A guideline, and it was GLP. However, the influent sewage was dosed intermittently, not continuously as required in a CAS test. Moreover, the effluent was not preserved when sampled, permitting additional biodegradation after exiting the CAS unit.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on source and properties of surface water:

This section refers to influent fed to CAS units.
- Details on collection: raw sewage (mostly domestic) collected from Bocholt, Belgium, enriched with synthetic sewage
- Storage conditions: 4 deg C
- Storage length: no data
- Temperature (°C) at time of collection: no data
- pH at time of collection: no data
- Electrical conductivity: no data
- Redox potential (mv) initial/final: no data
- Oxygen concentration (mg/L) initial/final: no data
- Hardness (CaCO3): no data
- Dissolved organic carbon (%): no data
- Biomass: COD from collected influent ranged between 220 and 480 mg/L; goal with volumes of synthetic sewage and sewage from Bocholt was to acheive a COD value between 260 and 430 mg/L.
- Water filtered: no data
- Type and size of filter used, if any: N/A

Details on source and properties of sediment:

N/A

Details on inoculum:

- Source of inoculum/activated sludge: from the aerators of a treatment plant dealing primarily with domestic sewage (Bocholt)
- Laboratory culture: no data
- Method of cultivation: no data
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: no data
- Pretreatment: The activated sludge was acclimated to the non-radiolabelled test substance (E-5138.01) for 3 weeks (from test day 0 until day 24).
- Concentration of sludge: Suspended solids content of the inoculum was 3.88 g/l.
- Initial cell/biomass concentration: no data

Duration of test (contact time):

21 d

Initial conc.:

110 µg/L

Based on:

other: specific activity (Unit 1)

Initial conc.:

384 µg/L

Based on:

other: specific activity (Unit 2)

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: The outlet of the separation vessel (used to collect wasted sludge) was fixed such that the volume contained in the aeration vessel was 3 L.
- Composition of medium: The composition of the synthetic sewage was as follows: peptone (160 mg/L); meat extract (110 mg/L); urea (30 mg/L); NaCl (7 mg/L); CaCl2.2H2O (4 mg/L); MgSO4.7H2O (2 mg/L); and K2HPO4 (28 mg/L).
- Additional substrate: no data
- Solubilising agent (type and concentration if used): N/A
- Test temperature: Temperature ranged 18-24 °C in the aeration vessels. Temperature in room ranged 15-28 °C.
- pH: ranged between 6-8
- pH adjusted: no data
- CEC (meq/100 g): no data
- Aeration of dilution water: no, aeration of aeration vessel - Dissolved oxygen concentration was >= 6.5 mg DO/l during test period.
- Suspended solids concentration: 3.88 g/L (inoculum)
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: no data
- Other: N/A

TEST SYSTEM
- Culturing apparatus: The experimental apparatus consisted of a storage vessel for the sewage, a storage vessel for the stock solution of the test substance, dosing pumps, an aeration vessel, a separator, an air-lift pump to recycle the activated sludge, and a vessel for collecting the treated effluent.
- Number of culture flasks/concentration: 1 flask/ concentration and two trial systems
- Method used to create aerobic conditions: The rate of aeration was regulated so that the contents of the aerator were kept constantly in suspension while the dissolved oxygen concentration was always above 3 mg/L.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: COD: Pocket-Photometer LASA 3 and LSC
- Test performed in closed vessels due to significant volatility of test substance: no, performed in closed system to collect evolved 14CO2
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The aerator and the separator were closed, and two absorber bottles, each containing 500 mL ethanolamine, were linked to the headspace of the aerator to collect the evolved 14CO2.
- Other: The test solution was introduced into the aeration vessel at 1000 mL/day and the overall influent flow rate was 9000 mL/day. The inoculum was introduced to the aeration vessel at a concentration of 2-2.5 g/L. Sewage was added in a discontinuous matter (4 hours of sewage addition, 4 hours no sewage addition). (Note: in a typical continuous activated sludge test, the sewage is added continuously.) The sludge retention time (SRT) was 10 days and was regulated by wastage of 350 mL/day of the mixed liquor. The hydraulic retention time (HRT) was 8 hours. The effluent from the separator was collected in a vessel for 17 hours after which sampling was performed. (Note: because the effluent was not preserved upon collection, the evaluation of parent and metabolites in the effluent is not reliable.) The COD of the CAS influent ranged from 360-480 mg/L (nominal).

SAMPLING
- Sampling frequency: The sampling frequency for LSC was 5 times per week during the radiolabelled period. The sampling frequency of the effluent for COD analysis was three times per week during the entire testing period. The COD of the sewage was measured whenever the CAS influent solution was renewed.
- Sampling method: For LSC, all samples were collected and analyzed in duplicate. For measurement of radiolabelled test substance in the effluent, duplicate samples of total effluent (2 mL) were analyzed. For the supernatant, 100 mL of the effluent was centrifuged and 2 mL of the supernatant was analyzed. Dissolved CO2 was determined by addition of 1 mL concentrated HCl to 10 mL total effluent in a biometer flask containing 3 mL ethanolamine and 1 mL samples of ethanolamine was analyzed 24 hours after addition of the acid. In the sludge, 2 mL samples of the mixed liquor was sampled. In addition, 100 mL sludge was sampled and centrifuged and 2 mL samples of the supernatant were taken and analyzed. The dissolved CO2 was determined by addition of 1 mL concentrated HCl to 10 mL supernatant in a biometer flask containing 3 mL ethanolamine and 1 mL samples of ethanolamine were analyzed after addition of the acid. Duplicate 2 mL samples were taken from each CO2 trap bottle. The first bottle of the CO2 trap was renewed each week and the second bottle was not renewed.
- Sterility check if applicable: no sterility check was performed
- Sample storage before analysis: Test substance solution and effluent samples were poured into polyethylene bottles and stored in a freezer until analysis. Activated sludge samples were poured into centrifuge tubes and were centrifuged for 10 min at 9000 g. The supernatants and residues were poured into polyethylene bottles and stored in a freezer.
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: none
- Abiotic sterile control: none
- Toxicity control: none
- Other: N/A

STATISTICAL METHODS:
- Statistical evaluation was performed on the results of overall removal, removal of biodegradation (mineralization), removal by sorption, overall organic removal and mass balance. Statistical evaluation was also performed on the COD removal. The reported statistical parameters are: the number of samples (n); number of outlier; the means and standard deviations; and the 95% confidence limits.

Reference substance:

not required

Test performance:

- The room temperature range was 15-28 deg C during the test period. The temperature in the activated sludge reactor was in the range of 18.7-23.8 deg C fot test unit 1, and 18.4-23.8 for test unit 2 during the test period.
- The dissolved oxygen concentration was >= 6.5 mg DO/L during the test period.
- The pH of the mixed liquor ranged between 6.02 and 7.40.
- The mixed liquor suspended solids (MLSS) ranged between 1-3 g/L, except on 1995-09-22, when a value of 4.22 was recorded in 4.22.
- The COD values in the influent ranged from 360-480 ug/L.

% Degr.:

> 99.9

Parameter:

other: % removal of parent

Sampling time:

21 d

Remarks on result:

other: Unit 1. Based on TLC-RAD analysis of effluent.

% Degr.:

99.8

Parameter:

other: % removal of parent

Sampling time:

21 d

Remarks on result:

other: Unit 2. Based on TLC-RAD analysis of effluent.

Transformation products:

yes

Details on transformation products:

By TLC-RAD, a total of 5 metabolites ("intermediates") of AO were observed, along with the parent compound. The chemical identity of each metabolite was not ascertained.

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not specified

Details on results:

By TLC-RAD, a total of 5 metabolites ("intermediates") of AO were observed, along with the parent compound. The chemical identity of each metabolite was not ascertained.

Additional Results The residual radioactivity in the MLSS was determined by LSC and averaged 490 and 1353 ug/L for test unit 1 and 2 respectively. After centrifugation of the MLSS, LSC was done on the liquid and the solid phase: for unit 1, 3% of the radiolabel was found in the liquid phase, 97% in the solid phase. For unit 2: 7% was found in the liquid phase, 93% in the solid phase. These results were confirmed by the LSC results of the solvent extracts of the liquid and solid phase from the MLSS after centrifugation: 91 % of the radioactivity was found on the solids for unit 1 and 90% of the radioactivity was found on the solids for unit 2. The MLSS were separated into a liquid and a solid fraction by centrifugation. The solid fractions were extracted and quantified by LSC. Parts of the remaining solids were combusted. The distribution of the radiolabel was 7% in the extractable phase and 93% on the solids. Compared to the LSC results of the solids, 95% of the radiolabel was recovered after extraction and combustion. The radiochemical speciation of the MLSS was determined separately on the liquid solid fractions. Both phases were separated by centrifugation and then, after lyophilisation and extraction, quantified by TLC-RAD. The results are shown below (expressed as equivalent amine oxide).   The speciation in the ML-extract of the MLSS from unit 1 (ug/L)   intermediate 1           0.04 intermediate 2           0.57 intermediate 3           0.06 intermediate 4           < 0.13 parent                        0.16 intermediate 5           0.38   The speciation in the ML-extract of the MLSS from unit 2 (ug/L)   intermediate 1           2.23 intermediate 2           0.83 intermediate 3           4.13 intermediate 4           < 0.13 parent                        1.07 intermediate 5           < 0.13   The speciation of the SS-extract of the MLSS from unit 1 (ug/g dry sludge)   intermediate 1           1 intermediate 2           1 intermediate 3           1 intermediate 4           2 parent                       2 intermediate 5           7

Validity criteria fulfilled:

yes

Remarks:

(With the exceptions that the CAS units were not continuously fed sewage, and the effluents were not adequately preserved.)

Conclusions:

A continuous activated sludge study of C12/C14 Amine Oxide, with radiochemical tracer, demonstrated a removal of >99.9% of parent (at 110 ug/L influent), and a removal of 99.8% (at 384 ug/L influent). These results are based on the TLC-RAD analysis of the effluent. Methodological limitations in the study make these results unreliable.

Executive summary:

The biodegradation of C12/C14 amine oxide (AO) was evaluated in a continuous activated sludge (CAS) test. The sludge and sewage were from a municipal sewage treatment plant receiving predominantly domestic waste. The CAS units were dosed with 14C-dodecyl amine oxide. After the CAS units stabilized, radioactivity in the effluent, evolved as CO2, and sorbed to solids was measured. Parent AO and metabolites were measured in effluent and MLSS. Effluent from the CAS units was also used in a subsequent river water die away test. The inoculum was acclimated to the test substance for 3 weeks prior to testing. Separate CAS units were operated at concentrations of 110 ug/L and 394 ug/L (as active ingredient AO). A radiolabelled 14C tracer was used to monitor mineralization and removal. In principle, the study followed the OECD 303A guideline, and it was GLP. However, the influent sewage was fed intermittently, not continuously as is typical in a CAS test. More importantly, the effluent was not adequately preserved when sampled, permitting additional biodegradation after exiting the CAS unit. The results from this study, specifically on removal of parent and formation of metabolites, are unreliable, due to the methodological limitations. Samples from the CAS test (effluent, etc.) were analyzed at Lisec (where the test was conducted), and at P&G (the sponsor). Lisec and P&G measured total radioactivity, and those results are reported in a separate robust summary. P&G also measured parent and metabolites by TLC-RAD, and those results are reported in this robust summary. Based on parent and metabolites in the effluent, the removal of parent AO was >99.9% (unit 1), and 99.8% (unit 2). A subsequent river water dieaway study of AO demonstrated further mineralization of the parent in the river water matrix.

Reference 6

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-08-17 to 1995-10-18 (DDAO CAS Study), and 1995-11-6 to 1995-12-6 (14C-DDAO Dieaway Study)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

In principle, the study followed the OECD 303A guideline, and it was GLP. However, the influent sewage was dosed intermittently, not continuously as required in a CAS test. Moreover, the effluent was not adequately preserved when sampled, permitting additional biodegradation after exiting the CAS unit.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Deviations:

yes

Remarks:

Treatment. A: Activated Sludge Units) yes (Discontinuous addition of sewage influent to the CAS unit (not continuous); inadequate preservation of effluent before analysis.)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on source and properties of surface water:

This section refers to influent fed to CAS units.
- Details on collection: raw sewage (mostly domestic) collected from Bocholt, Belgium, enriched with synthetic sewage
- Storage conditions: 4 deg C
- Storage length: no data
- Temperature (°C) at time of collection: no data
- pH at time of collection: no data
- Electrical conductivity: no data
- Redox potential (mv) initial/final: no data
- Oxygen concentration (mg/L) initial/final: no data
- Hardness (CaCO3): no data
- Dissolved organic carbon (%): no data
- Biomass: COD from collected influent ranged between 220 and 480 mg/L; goal with volumes of synthetic sewage and sewage from Bocholt was to acheive a COD value between 260 and 430 mg/L.
- Water filtered: no data
- Type and size of filter used, if any: N/A

Details on source and properties of sediment:

N/A

Details on inoculum:

- Source of inoculum/activated sludge: from the aerators of a treatment plant dealing primarily with domestic sewage (Bocholt)
- Laboratory culture: no data
- Method of cultivation: no data
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: no data
- Pretreatment: The activated sludge was acclimated to the non-radiolabelled test substance (E-5138.01) for 3 weeks (from test day 0 until day 24).
- Concentration of sludge: Suspended solids content of the inoculum was 3.88 g/l.
- Initial cell/biomass concentration: no data

Duration of test (contact time):

21 d

Initial conc.:

110 µg/L

Based on:

other: based on specific activity (Unit 1)

Initial conc.:

384 µg/L

Based on:

other: based on specific activity (Unit 2)

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: The outlet of the separation vessel (used to collect wasted sludge) was fixed such that the volume contained in the aeration vessel was 3 L.
- Composition of medium: The composition of the synthetic sewage was as follows: peptone (160 mg/L); meat extract (110 mg/L); urea (30 mg/L); NaCl (7 mg/L); CaCl2.2H2O (4 mg/L); MgSO4.7H2O (2 mg/L); and K2HPO4 (28 mg/L).
- Additional substrate: no data
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 18-24 °C (in the aeration vessels).
- pH: ranged between 6-8
- pH adjusted: no data
- CEC (meq/100 g): no data
- Aeration of dilution water: no, aeration of aeration vessel - Dissolved oxygen concentration was >= 6.5 mg DO/l during test period.
- Suspended solids concentration: 3.88 g/L (inoculum)
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: no data
- Other: N/A

TEST SYSTEM
- Culturing apparatus: The experimental apparatus consisted of a storage vessel for the sewage, a storage vessel for the stock solution of the test substance, dosing pumps, an aeration vessel, a separator, an air-lift pump to recycle the activated sludge, and a vessel for collecting the treated effluent.
- Number of culture flasks/concentration: 1 flask/ concentration and two trial systems
- Method used to create aerobic conditions: The rate of aeration was regulated so that the contents of the aerator were kept constantly in suspension while the dissolved oxygen concentration was always above 3 mg/L.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: COD: Pocket-Photometer LASA 3 and LSC
- Test performed in closed vessels due to significant volatility of test substance: no, performed in closed system to collect evolved 14CO2
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The aerator and the separator were closed, and two absorber bottles, each containing 500 mL ethanolamine, were linked to the headspace of the aerator to collect the evolved 14CO2.
- Other: The test solution was introduced into the aeration vessel at 1000 mL/day and the overall influent flow rate was 9000 mL/day. The inoculum was introduced to the aeration vessel at a concentration of 2-2.5 g/L. Sewage was added in a discontinuous matter (4 hours of sewage addition, 4 hours no sewage addition). (Note: in a typical continuous activated sludge test, the sewage is added continuously.) The sludge retention time (SRT) was 10 days and was regulated by wastage of 350 mL/day of the mixed liquor. The hydraulic retention time (HRT) was 8 hours. The effluent from the separator was collected in a vessel for 17 hours, mixed, and then sampled. (Note: because the effluent was not preserved upon collection, biodegradation of the test material may have continued until the effluent was sampled.) The COD of the CAS influent ranged from 360-480 mg/L (nominal).

SAMPLING
- Sampling frequency: The sampling frequency for LSC was 5 times per week during the radiolabelled period. The sampling frequency of the effluent for COD analysis was three times per week during the entire testing period. The COD of the sewage was measured whenever the CAS influent solution was renewed.
- Sampling method: For LSC, all samples were collected and analyzed in duplicate. For measurement of radiolabelled test substance in the effluent, duplicate samples of total effluent (2 mL) were analyzed. For the supernatant, 100 mL of the effluent was centrifuged and 2 mL of the supernatant was analyzed. Dissolved CO2 was determined by addition of 1 mL concentrated HCl to 10 mL total effluent in a biometer flask containing 3 mL ethanolamine and 1 mL samples of ethanolamine was analyzed 24 hours after addition of the acid. In the sludge, 2 mL samples of the mixed liquor was sampled. In addition, 100 mL sludge was sampled and centrifuged and 2 mL samples of the supernatant were taken and analyzed. The dissolved CO2 was determined by addition of 1 mL concentrated HCl to 10 mL supernatant in a biometer flask containing 3 mL ethanolamine and 1 mL samples of ethanolamine were analyzed after addition of the acid. Duplicate 2 mL samples were taken from each CO2 trap bottle. The first bottle of the CO2 trap was renewed each week and the second bottle was not renewed.
- Sterility check if applicable: no sterility check was performed
- Sample storage before analysis: Test substance solution and effluent samples were poured into polyethylene bottles and stored in a freezer until analysis. Activated sludge samples were poured into centrifuge tubes and were centrifuged for 10 min at 9000 g. The supernatants and residues were poured into polyethylene bottles and stored in a freezer.
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: none
- Abiotic sterile control: none
- Toxicity control: none
- Other: N/A

STATISTICAL METHODS:
- Statistical evaluation was performed on the results of overall removal, removal of biodegradation (mineralization), removal by sorption, overall organic removal and mass balance. Statistical evaluation was also performed on the COD removal. The reported statistical parameters are: the number of samples (n); number of outlier; the means and standard deviations; and the 95% confidence limits.

Reference substance:

not required

Test performance:

- The room temperature range was 15-28 deg C during the test period. The temperature in the activated sludge reactor was in the range of 18.7-23.8 deg C fot test unit 1, and 18.4-23.8 for test unit 2 during the test period.
- The dissolved oxygen concentration was >= 6.5 mg DO/L during the test period.
- The pH of the mixed liquor ranged between 6.02 and 7.40.
- The mixed liquor suspended solids (MLSS) ranged between 1-3 g/L, except on 1995-09-22, when a value of 4.22 was recorded in 4.22.
- The COD values in the influent ranged from 360-480 ug/L.

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: Mass balance in CAS Unit 1 (dosed with 110 ug/L test substance) was 100.2%. Mass balance in CAS Unit 2 (dosed with 384 ug/L test substance) was 97.5%.

% Degr.:

86.7

St. dev.:

3

Parameter:

other: Mass balance in CAS Unit 1 (dosed with 110 ug/L test substance) was 100.2%. Mass balance in CAS Unit 2 (dosed with 384 ug/L test substance) was 97.5%.

Sampling time:

21 d

Remarks on result:

other: Unit 1. Measured at Lisec. Based on total radioactivity in effluent, minus dissolved CO2 in effluent.

% Degr.:

92.6

Parameter:

other: % overall AO removal

Sampling time:

21 d

Remarks on result:

other: Unit 1. Measured at P&G. Based on total radioactivity in effluent, minus dissolved CO2 in effluent.

% Degr.:

75

St. dev.:

8.4

Parameter:

CO2 evolution

Sampling time:

21 d

Remarks on result:

other: Unit 1. Measured at Lisec.

% Degr.:

16.1

St. dev.:

4.5

Parameter:

other: % removal by sorption to MLSS

Sampling time:

21 d

Remarks on result:

other: Unit 1. Measured at Lisec.

% Degr.:

79.4

St. dev.:

7.1

Parameter:

other: % overall AO removal

Sampling time:

21 d

Remarks on result:

other: Unit 2. Measured at Lisec. Based on total radioactivity in effluent, minus dissolved CO2 in effluent.

% Degr.:

86

St. dev.:

5

Parameter:

other: % overall AO removal

Sampling time:

21 d

Remarks on result:

other: Unit 2. Measured at P&G. Based on total radioactivity in effluent, minus dissolved CO2 in effluent.

% Degr.:

69.9

St. dev.:

7.7

Parameter:

CO2 evolution

Sampling time:

21 d

Remarks on result:

other: Unit 2. Measured at Lisec

% Degr.:

12.2

St. dev.:

3.1

Parameter:

other: % removal by sorption to MLSS

Sampling time:

21 d

Remarks on result:

other: Unit 2. Measured at Lisec.

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Validity criteria fulfilled:

yes

Remarks:

(With the exceptions that the CAS units were not continuously fed sewage, and the effluents were not adequately preserved.)

Conclusions:

(With the exceptions that the CAS units were not continuously fed sewage, and the effluents were not adequately preserved.)

Executive summary:

The biodegradation of C12/C14 amine oxide (AO) was evaluated in a continuous activated sludge (CAS) test. The sludge and sewage were from a municipal sewage treatment plant receiving predominantly domestic waste. The CAS units were dosed with 14C-dodecyl amine oxide. After the CAS units stabilized, radioactivity in the effluent, evolved as CO2, and sorbed to solids was measured. Effluent from the CAS units was also used in a subsequent river water die away test. The inoculum was acclimated to the test substance for 3 weeks prior to testing. Separate CAS units were operated at concentrations of 110 ug/L and 394 ug/L (as active ingredient AO). A radiolabelled 14C tracer was used to monitor mineralization and removal to solids and biomass.. In principle, the study followed the OECD 303A guideline, and it was GLP. However, the influent sewage was fed intermittently, not continuously as is typical in a CAS test. Moreover, the effluent was not adequately preserved when sampled, permitting additional biodegradation after exiting the CAS unit. The results from this study are reliable, but restricted, due to the methodological limitations. Samples from the CAS test (effluent, etc.) were analyzed at Lisec (where the test was conducted), and at P&G (the sponsor). Lisec and P&G measured total radioactivity, and those results are reported in this robust summary. P&G also measured parent and metabolites (those results reported in a separate robust summary). Based on total radioactivity in the effluent, minus the dissolved 14CO2 in the effluent, the removal of AO was 87 -93% (unit 1), and 79 -86% (unit 2). The mineralization of the test substance was 75% (unit 1) and 70% (unit 2). The sorption to MLSS was 16% (unit 1) and 12% (unit 2). Mass balance was 100% (unit 1) and 98% (unit 2). A subsequent river water dieaway study of AO demonstrated further mineralization of the parent in the river water matrix.

Reference 7

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is readily biodegradable

Description of key information

In a Continuous activated sludge (CAS) test performed using C12-14 AO, removal of parent amine oxide was > 99.8 % in both units. In a monitoring study performed in the Netherlands, the influent and effluent concentrations of C12 and C14 amine oxide was measured in six municipal sewage treatment plants. The BOD removal was 96-98 %. On the basis of these studies, a removal rate in STP of 98 % is proposed for modelling. In a river water die away study the half life of amine oxide was determined to be 3.2 days. In a biodegradation test performed using anaerobic digester sludge the rate constant for mineralization of the amine oxide was 1.32/day. The results of these studies are read across to C14-16 AO on the basis of the equivalence in chemical structure.

Key value for chemical safety assessment

Half-life in freshwater:

3.2 d

at the temperature of:

20 °C

Additional information

No specific test data are available for C14 -16 AO, however a number of studies have been performed with C12 -14 AO. The findings in these studies are read across to C14 -16 AO on the basis of the equivalence in chemical structure. The biodegradation of C12/C14 amine oxide (AO) was evaluated in a continuous activated sludge (CAS) test [Lisec (1995)]. The sludge and sewage were from a municipal sewage treatment plant receiving predominantly domestic waste. The CAS units were dosed with 14C-dodecyl amine oxide. After the CAS units stabilized, radioactivity in the effluent, evolved as CO2, and sorbed to solids was measured. Parent AO and metabolites were measured in effluent and mixed liquor suspended solids (MLSS). Effluent from the CAS units was also used in a subsequent river water die away test.

The inoculum was acclimated to the test substance for 3 weeks prior to testing. Separate CAS units were operated at concentrations of 110 ug/L and 394 ug/L (as active ingredient AO). A radiolabelled 14C tracer was used to monitor mineralization and removal.

In principle, the study followed the OECD 303A guideline, and it was GLP. However, the influent sewage was fed intermittently, not continuously as is typical in a CAS test. More importantly, the effluent was not adequately preserved when sampled, permitting additional biodegradation after exiting the CAS unit. Overall, the results of this study are considered to be reliable, with the specific exception of the data on the removal of the parent and formation of metabolites, which are considered to be unreliable.

Samples from the CAS test (effluent, etc.) were analyzed at the laboratory where the test was conducted and also at the laboratories of the sponsor company. The test laboratory and the sponsor both measured total radioactivity (results summarised below). Based on total radioactivity in the effluent, minus the dissolved 14CO2 in the effluent, the removal of AO was 87 -93% (unit 1), and 79 -86% (unit 2). The mineralization of the test substance was 75% (unit 1) and 70% (unit 2). The sorption to MLSS was 16% (unit 1) and 12% (unit 2). Mass balance was 100% (unit 1) and 98% (unit 2). The sponsor also measured parent and metabolites by TLC-RAD. Based on parent and metabolites in the effluent, the removal of parent AO was >99.9% (unit 1), and 99.8% (unit 2).

The removal of C12/14 amine oxide from wastewater by sewage treatment was determined in a monitoring study in The Netherlands between May and July, 1996 [Debaere G (1996c)]. Six municipal activated sludge treatment plants in The Netherlands were included. The concentration of C12/14 amine oxide was determined in influent and effluent samples. Samples (raw and treated sewage) were collected from the six municipal activated sludge sewage treatment plants (STP) over three consecutive days. The samples were analysed by FI/MS/MS for both C12 and C14 Amine oxide. The removal of amine oxide during activated sludge sewage treatment was >95 to >99% at the six plants. The level of Amine oxide in effluent was below detection in all effluent samples (<0.43 µg/L). The amount of Amine oxide in influent ranged between 9-130 ug/L. The BOD removal at the six plants was 96 to >98%.

Based on the results of the CAS test (removal of parent AO >99.8 % in both units) and the removal of AO from wastewater by sewage treatment seen in the Netherlands monitoring study (BOD 96 to > 98 %) it is considered to be justified to use a removal rate of 98 % when evaluating exposure to the aquatic compartment from release of AO to the environment via STP.

The biodegradation of dodecyl amine oxide (C12 AO) was evaluated in a River-Water Die Away study according to the OECD 314D guidelines [Debaere G (1996)]. The test system consisted of river water, effluent from 2 CAS units, and a small amount of activated sludge from a treatment plant handling primarily domestic sewage. The test period was 14 days, and 14CO2 production and radiochemistry were measured. The river water die away (RDA) test was performed in duplicate: RDA test 1 used effluent from CAS unit 1, and RDA test 2 used effluent from CAS unit 2. RDA test 1 was dosed with 2 ug/L test substance (from the effluent from CAS unit 1). RDA test 2 was dosed with 1 ug/L test substance, and 2.9 ug/L metabolites of parent (from the effluent from CAS unit 2).

After 14 days, the total 14CO2 production in the river die away study was 43% for RDA test 1, and 63% for test unit 2. The half-life of mineralization was 4.3 days in RDA 1, and 2.1 days in RDA 2. After 14 days, loss of parent was 61% in RDA test 1. The % loss of parent in unit 2 was unreliable (7%), because of the presence of significant metabolites at the start in RDA test 2. After 14 days, the amount of parent compound was 0.78 ug/L in RDA test 1, and 0.92 ug/L in RDA test 2. Intermediates were mineralized to below detection limits of the TLC-RAD method (<0.33 ug/L).

The radiochemical mass balances averaged 87 and 99% for RDA tests 1 and 2 respectively. After 14 days in RDA test 1, 43% of the radioactivity was CO2, 39% was parent, and 9% was associated with the solids (total=91%). After 14 days in RDA test 2, 63% of the radioactivity was CO2, 24% was parent, and 24% was associated with the solids (total=111%). Based on the results of the two tests, the half-life in surface water is 3.2 days (the average of 4.3 and 2.1).

A simulation of the mineralization of C12 alkyl amine oxide in anaerobic digester sludge was conducted in accordance with OECD 314C guideline [Edwards DE (1996a)]. A solution of radiolabeled and non-radiolabeled amine oxide was tested at 1 mg/L. The radiolabel (14C) was on the methylene groups (CH2) of the alkyl side-chain.The inoculum was acclimated anaerobic digester sludge harvested from a bench scale anaerobic acclimation reactor. The test treatments were measured in duplicate. The evolution of both 14C-CO2 and 14C-methane was measured. The mean cumulative percent of theoretical % 14C-gas produced was 75.5.The rate constant for mineralization of amine oxide in anaerobic digester sludge was 0.055/hour (equivalent to 1.32/day).

According to Annex IX Section 9.2.1.4 sediment simulation testing is required for substances with a high potential for adsorption to sediment. However column 2 of section 9.2.1.3 states that the study need not be conducted if the substance is readily biodegradable, or if direct and indirect exposure of sediment is unlikely. The substance is readily biodegradable and hence the study is scientifically unjustified.