N,N-dimethyldecylamine N-oxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Hildesheim waste water treatment plant. This WWTP receives primarily domestic wastewater
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: The sludge was aerated until use
- Storage length: not recorded
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration: 35000 colony-forming units/mL
- Water filtered: yes
- Type and size of filter used, if any: Folded paper filter

Duration of test (contact time):

28 d

Initial conc.:

150 mg/L

Based on:

test mat.

Initial conc.:

45.9 mg/L

Based on:

act. ingr.

Initial conc.:

28.95 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was made according to the OECD 301E guideline.
- Additional substrate: not applicable
- Solubilising agent: non used
- Test temperature: 22+/-2 deg C
- pH: 7.4
- pH adjusted: no
- CEC (meq/100 g): not applicable
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: 2000 mL Erlenmeyer flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: TOC analyser - TOCOR2, Fa. Maihak
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no data

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:

Reference substance:

acetic acid, sodium salt

Remarks:

(Test concentration 130 mg/L; ThTOC in test solution 37.3 mgC/L)

Preliminary study:

Not applicable

Test performance:

No data

Parameter:

% degradation (DOC removal)

Value:

18

Sampling time:

5 d

Remarks on result:

other: mean of 2 replicates

Parameter:

% degradation (DOC removal)

Value:

72

Sampling time:

7 d

Remarks on result:

other: mean of 2 replicates

Parameter:

% degradation (DOC removal)

Value:

97

Sampling time:

28 d

Remarks on result:

other: mean of 2 replicates

Results with reference substance:

The reference substance met the validity criteria for the test (>60 % biodegradation within 14 d and within the 10 d window). Pass level of 60 % reached by Day 5 (Day 1 44%, Day 5 93 %, Day 28 96 %).

Table 1: DOC results for control and reference substance

Day	Control			Reference substance (130 mg/L)
	DOC (mg C/L)			DOC (mg C/L)		(%)
	K1	K2	Av.	R	Nett
0	12.67	17.70	15.19	49.34	34.15	-
1	0.99	1.48	1.24	20.33	19.09	44
5	0.59	1.22	0.91	3.24	2.33	93
7	1.43	2.14	1.79	4.83	3.04	91
14	3.09	1.92	2.51	3.26	0.75	98
21	2.81	3.29	3.05	2.60	0	100
28	1.44	0.76	1.10	2.48	1.38	96

 

Table 2: DOC results for test substance

Day	Test substance (150 mg/L)
	DOC (mg C/L)				(%)
	P1	P2	Av	Nett
0	40.6	37.9	39.25	24.06	-
1	34.67	28.38	31.53	30.29	0
5	20.62	20.58	20.60	19.69	18
7	8.28	8.75	8.52	6.73	72
14	5.20	5.31	5.26	2.75	89
21	6.94	2.11	4.53	1.48	94
28	2.05	1.76	1.91	0.81	97

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Executive summary:

A ready biodegradation test was performed according to OECD TG 301E under GLP using C10 AO. The substance was tested at 45.9 mg AO/L (28.95 mg C/L). The test treatment and control were tested in duplicate, whilst a single reference control was used. Eighteen percent biodegradation of the substance was reached at Day 5 and 72 % biodegradation was reached at Day 7. Hence the substance met the 10-day window requirement for ready biodegradability. The final level of biodegradability at 28 days was 97 %.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 23, 2006 to September 20, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

However, this laboratory is in the process of attaining GLP status. This study was conducted in principle in accordance with GLPs.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from Fairfield waste water treatment plant (Fairfield, Ohio) on August 22, 2006. This WWTP receives primarily domestic wastewater.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: Sludge was aerated until use
- Storage length: Not reported
- Preparation of inoculum for exposure: The activated sludge solids were centrifuged for 20 minutes at 5000 rpm and the supernatant decanted. The solids were resuspended in media and homogenized in a blender for 1 min. The solids were washed a second time as described above and the TSS (total suspended solids) measured. Sufficient inoculum was added to the media to obtain solids concentration of 10 mg/L. This mixture was adjusted to pH 7.0 and aerated overnight with CO2 free air.
- Pretreatment: None
- Concentration of sludge: 10 mg solids/L
- Initial cell/biomass concentration: Not reported
- Water filtered: Not applicable
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

act. ingr.

Initial conc.:

14.25 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 301 B guideline.
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): None used
- Test temperature: 22±1°C
- pH: 7
- pH adjusted: Not reported
- CEC (meq/100 g): Not applicable
- Aeration of dilution water: The test solutions (mineral media plus inoculum) were aerated overnight with CO2-free air before the addition of test substance or reference
- Suspended solids concentration: 10 mg solids/L
- Continuous darkness: Not reported
- Other: None

TEST SYSTEM
- Culturing apparatus: 1L bottle
- Number of culture flasks/concentration: Three
- Method used to create aerobic conditions: Continuous aeration with CO2-free air.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: Electrolytic respirometer (Coordinated Environmental Services Ltd., UK) was used for measuring CO2. The Soluble organic carbon (SOC) was determined using a Shimadzu Total Organic Carbon Analyzer.
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Details of trap for CO2: A conductivity probe immersed in 1% NaOH is used to measure the production of CO2. The base reservoirs for the conductivity probes were prepared one day prior to test initiation and allowed to equilibrate at 22° C. NaOH was degassed via filtration, precisely measured (18 mL) into the base cups and subsequently attached to the probes.
- Other:
- Test set up: Each sample assembly consisted of sample vessel with a conductivity probe, an electrolysis chamber (with cathode and anode electrodes in a saturated solution of CuSO4) and an empty bottle served as a reference vessel for the pressure transducer pressure transducer. All three components comprised the sensory assembly and were attached to the sample vessel.
- Test vessel setup: 20 mL of stock solution was added to each replicate, along with inoculum and media to reach a total volume of one liter in each test vessel.

SAMPLING
- Sampling frequency: Sampling was performed automatically via the integrated computer software and was programmed for a 12 h sampling interval following a 1 h stabilization period.
- Sampling method: Automated sampling
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not supplied
- Other: None

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, there were 3 replicates of the blank (no inoculum present).
- Abiotic sterile control: No
- Toxicity control: No
- Procedure control: Yes

STATISTICAL METHODS: Not reported

Reference substance:

benzoic acid, sodium salt

Remarks:

tested at 20 mg/L (12.12 mg Carbon/L); ThOC: 58.3%; TOC of its stock solution: 606.1 mg C/L)

Preliminary study:

Not applicable

Test performance:

No data

Parameter:

% degradation (CO2 evolution)

Value:

13.96

St. dev.:

1.9

Sampling time:

3 d

Remarks on result:

other: Mean value of 3 replicates, indicating start of 10 d window criterion

Parameter:

% degradation (CO2 evolution)

Value:

61.67

St. dev.:

1.94

Sampling time:

9 d

Remarks on result:

other: Mean value of 3 replicates, indicating that the 10 d window criterion was met

Parameter:

% degradation (CO2 evolution)

Value:

91.22

St. dev.:

2.76

Sampling time:

28 d

Remarks on result:

other: Mean value of 3 replicates, indicating final percentage of degradation

Details on results:

- Final SOC in (mg/L) was as found to be 0.5, 0.5 and 1.5 for replicate 1, 2 and 3 respectively. The initial TOC was 14.25 mg/L.
- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’

Results with reference substance:

- The Reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window. Pass level of >60% reached after Day 5 in all replicates. (Mean values of all replicates on Day 1 = 17.4%, Day 5 = 61.6% and Day 28 = 93.6% TCO2).
For details on individual replicate results, please refer to 'Table 2' in the ‘Any other information on results incl. tables’.

- Final mg Soluble Organic Carbon (SOC)/L were found to be 0.2, 0.2 and 0.3 mg/L in replicate 1, 2 and 3. The initial TOC was 12.12 mg/L.

Table 1. Ready biodegradation test of amine oxide 1095 (SWF-866 -041) (study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2	Replicate 3
3	13.3	12.48	10.45
8	-	-	60.67
9	60.58	60.51	-
28	88.42	91.31	93.93

 

Table 2. Ready biodegradation test of reference substance (study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2	Replicate 3
1	16.73	18.65	16.73
5	62.60	63.39	61.34
28	92.52	96.74	91.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

C10 Amine oxide (C10 AO) is readily biodegradable.

Executive summary:

A ready biodegradation test, following OECD 301B guideline, was conducted on C10 amine oxide. The substance was tested at 20 mg active/L (14.25 mg TOC/L) and the inoculum was 10 mg activated sludge solids/L. The test treatment, inoculum blank and reference control were measured in triplicate. The deviation between replicate treatments was <20% (met the validity criteria of the guideline). 

Ten percent biodegradation of the test material was reached at Day 3 and 60% biodegradation was reached at Day 9 in all replicates. At Day 3, the average biodegradation was 14% (±1.90), and at Day 9, the average biodegradation was 61.7% (±1.94). Hence the test material met the 10 d window requirement for ready biodegradability.

The final level of biodegradation at 28 days was 91.2% (±2.76).

The reference material, sodium benzoate, reached 10% biodegradation at Day 1 and >60% after Day 4. It met the validity criteria established in the guideline for a reference material.

This ready biodegradation test is classified as acceptable, and satisfies the guideline requirements for the OECD 301B biodegradation screening test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

The dilution water was the medium as prescribed by the test guideline, without ammonia, to minimise the consumption of oxygen for the nitrification process.

GLP compliance:

yes

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Source of inoculum/activated sludge: No data

Duration of test (contact time):

4 wk

Initial conc.:

6.8 mg/L

Based on:

not specified

Value:

93

Sampling time:

4 wk

Remarks on result:

other: At a concentration of 6.8 mg Aromox DMMCD-W/L.

Details on results:

According to the recommendations of the OECD, Aromox DMMCD-W should be regarded as readily biodegradable. In the toxicity control, the concentration of oxygen for the biodegradation of sodium acetate was not inhibited by Aromox DMMCD-W at concentrations up to 20.5 mg/L.

Parameter:

BOD5

Value:

4.7 other: mg/L

Parameter:

COD

Value:

5.05 other: mg/L

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

According to the recommendations of the OECD, Aromox DMMCD-W should be regarded as readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01-11-2000 to 29-11-2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

not specified

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Duration of test (contact time):

28 d

Initial conc.:

14.8 other: mg/L

Based on:

test mat.

Initial conc.:

22.7 other: % (m/m)

Based on:

other: TOC

Initial conc.:

0.8 other: mg CO2/mg substance

Based on:

other: ThCO2

Initial conc.:

36.9 other: mg CO2/3 L

Based on:

other: THCO2 in the test vessel.

Parameter:

% degradation (CO2 evolution)

Value:

80

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

2 d

Remarks on result:

other: Marked the beginning of biodegradation.

Details on results:

The test substance at a concentration of 15 mg/L reached a degree of degradation of 80 % after 28 days. The level of 10 % degradation (the beginning of degradation) was reached after 2 days. The 60 % level was passed after 10 days. Low test substance concentration was chosen because amine oxides may be inhibitory. The pass level for ready biodegradability, i.e. ≥ 60 % theoretical CO2 production within 10 days after the degree of degradation has reached 10 % (10-day window) was reached. The test substance is therefore readily biodegradable.

Summary:

	Genaminox LA	Reference substance (Sodium benzoate)
Lag time t1	3 days	1.5 days
Degradation time t2	13 days	9.5 days
Maximum level of degradation	80 %	89 %
Readily biodegradable (10 d window)	Yes	Yes

The reference substance reached the pass level of 60 % degradation within 14 days. The toxicity control did not reach a degree of degradation of ≥ 35 % within 14 days. Since the DOC was eliminated after 28 days it was supposed that there was a leak in the system and that therefore not the complete CO2 was trapped. The CO2 evolution in the inoculum blank was ≤ 40 mg/L in 28 days. The difference of extremes of replicate values of the removal of the test and at the end of the 10 day window should not be more than 20 %. This criterium is not applicable becuase the test substance was only tested in a single assay.

Validity criteria fulfilled:

yes

Remarks:

The reference substance reached the pass level of 60 % degradation within 14 days. The toxicity control did not reach a degree of degradation of ≥ 35 % within 14 days due to a probable leak in the system.

Interpretation of results:

readily biodegradable

Conclusions:

The test substance at a concentration of 15 mg/L reached a degree of degradation of 80 % after 28 days. The level of 10 % degradation (the beginning of degradation) was reached after 2 days. The 60 % level was passed after 10 days. Low test substance concentration was chosen because amine oxides may be inhibitory. The pass level for ready biodegradability, i.e. ≥ 60 % theoretical CO2 production within 10 days after the degree of degradation has reached 10 % (10-day window) was reached. The test substance is therefore readily biodegradable. The reference substance reached the pass level of 60 % degradation within 14 days. In a toxicity test, containing both the test substance and the reference substance, the degradation reached 12 %. Since the DOC was eliminated after 28 days it was supposed that there was a leak in the system and the complete CO2 was not trapped.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: German Standard Method 38 412

GLP compliance:

not specified

Remarks:

Study pre-dates GLP.

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge (adaptation not specified)

Duration of test (contact time):

28 d

Initial conc.:

2 g/L

Based on:

test mat.

Value:

86

Sampling time:

28 d

Remarks on result:

other: Meets 10-d window

Interpretation of results:

readily biodegradable

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05-01-2005 to 24-02-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Storage length: Used on the day of collection.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium and then was maintained on continuous aeration in the laboratoryat a temperature of 21 ºC. A sample of the activated sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may be present. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a washed sample of the activated sewage sludge by suction through pre-weighed GF/A filter paper (previously rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. The filter paper was then dried in an oven at approximately 105 ºC for at least an hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 2.1 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

73.2 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

other: mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The culture medium was that recommended in the OECD Guideline (see table).
- Solubilising agent: Not applicable
- Test temperature: 21 ºC
- pH: pH was determined on Day 28, prior to acidification. The pH of the test vesels was 7.4 to 7.5.
- pH adjusted: No
- Aeration: CO2 -free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Suspended solids concentration: 30 mg suspended solids/L.
- Continuous darkness: Yes


TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels each containing 3 L of solution.
- Number of culture flasks/concentration: Duplicate
- Method used to create aerobic conditions: Culture vessels were sealed and CO2-free air was bubbled through the solution at a rate of approximately 40 mL/min and stirred continuously by magnetic stirrer.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.


SAMPLING
- Sampling frequency: CO2 analysis: samples were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
- Sampling method: Samples were analysed for CO2 immediately, with the exception of samples from Day 12 and Day 18, which were stored at approximately -20ºC. These two samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that degradation of the test material met the 10-day window and therefore additional analyses were considered to be unnecessary.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser, an Ionics 1555B TOC analyser and Shimadzu TOC-VCSH TOC analyser. Samples (300, 50 or 40 μL) were injected into the Inorganic Carbon (IC) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 μL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis was carried out at 680 ºC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involved conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate and sodium carbonate in deionised water. Each analysis was carried out in triplicate.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate control consisting of inoculated culture medium.
- Other: Standard material (sodium benzoate) in duplicate in inoculated culture medium to give a final concentration of 10 mg carbon/L.

Reference substance:

other: Sodium benzoate

Preliminary study:

Preliminary investigational work carried out using the Activated Sludge Respiration Inhibition test (OECD Guideline No. 209) showed that the test material did not inhibit the respiration rate of the sewage sludge microorganisms at the concentration employed in the test. It was therefore considered acceptable to conduct the test at a concentration of 10 mg/L.

Test performance:

The validation criteria were fulfilled. Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicoty control vessel was a light brown dispersion with no undissoled standard materialor test material visible.

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

1 d

Parameter:

% degradation (CO2 evolution)

Value:

42

Sampling time:

2 d

Parameter:

% degradation (CO2 evolution)

Value:

72

Sampling time:

8 d

Parameter:

% degradation (CO2 evolution)

Value:

90

Sampling time:

28 d

Details on results:

The CO2 evolution in the control vessels on Day 28 was 22.47 mg/L.
The difference between the values for CO2 production at the end of the test for the repicate vessels was < 20 %.
The test material attained 90 % degradation after 28 days and satisfied the 10 day window.
The results of the inorganic carbon analysis of samples from the first absorber vessles on Day 29 showed an increase in all replicate vessels. These increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in a decrease in the percentage degradation value for the test material from 90 % in Day 28 to 89 % on day 29. The decrease in degradation obtained for the test materil on day 29 was considered due to the increases in inorganic carbon within the replicate control vessels being greater than those within the replicate test vessels.
The toxicity control attained 101 % degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the test.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry over of CO2 in the the second absorber vessels occurred.
Analysis of test media from the test material culture vessels on Days 0 and 28 for DOC gave percentage degradation values of 92 % and 89 % respectively for the test material replicates R 1 anad R2 and 95 % for the toxicity control. The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

Results with reference substance:

Sodium benzoate attained 114 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inroganic carbon in the first absorber vessels in Day 29 resulted in a decrease in the percentage degradation value for the standard material from 114 % on Day 28 to 110 % on Day 29. The decrease in degradation obtained for the standard material on Day 29 was considered to be due to the increases in inorganic carbon within the replicate control vessels being greater than those within the replicate standard material vessels. Degradation values in excess of 100 % were considered to be due to sampling/analytical variation.
Sodium benzoate attained 98 % and 99 % degradation respectively for replicates R1 and R2 calculated from the results of DOC analysis.

Percentage biodegradation values:

Day	% degradation sodium benzoate	% degradation test material	% degradation test material plus sodium benzoate toxicity control
0	0	0	0
1	29	6	30
2	61	42	68
3	86	58	78
6	89	58	88
8	87	72	89
10	86	81	96
14	84	90	99
16	89	93	99
20	88	90	101
22	97	85	97
24	101	91	106
27	105	91	106
28	114	90	101
29*	110	89	101

* Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 2.

Total and inorganic carbon values in the culture vessels on Day 0:

Test vessel	Total carbon* (mg/L)	Inorganic carbon* (mg/L)	IC/TC ratio (%)
Sodium benzoate 10 mg C/L R1	11.88	0.25	2
Sodium benzoate 10 mg C/L R2	10.73	0.41	4
Test material 10 mg C/L R1	7.81	-0.21	0
Test material 10 mg C/L R2	7.71	-0.65	0
Test material plus sodium benzoate toxicity control 20 mg C/L	17.21	-0.12	0

R1 - R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than control values.

Dissolved Organic Carbon (DOC) values in the culture vessles on Day 0 and 28:

DOC* Concentration
Test vessel	Day 0		Day 28
	mg C/L	% of nominal carbon content	mg C/L	% of initial carbon concentration	% degradation
Sodium benzoate 10 mg C/L R1	11.63	116	0.21	2	98
Sodium benzoate 10 mg C/L R2	10.32	103	0.11	1	99
Test material 10 mg C/L R1	8.02	80	065	8	92
Test material 10 mg C/L R2	8.36	84	0.93	11	89
Test material plus sodium benzoate toxicity control 20 mg C/L	17.33	87	0.83	5	95

R1 - R2 = Replicates 1 and 2

* Corrected for control values.

Validity criteria fulfilled:

yes

Remarks:

According to the OECD test guideline

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 90 % degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % degradation must be attained within 10 days of the degradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD guideline 301B.

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 23, 2006 to September 20, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

However, this laboratory is in the process of attaining GLP status. This study was conducted in principle in accordance with GLPs.

Specific details on test material used for the study:

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

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from Fairfield waste water treatment plant (Fairfield, Ohio) on August 22, 2006. This WWTP receives primarily domestic wastewater.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: Sludge was aerated until use
- Storage length: Not reported
- Preparation of inoculum for exposure: The activated sludge solids were centrifuged for 20 minutes at 5000 rpm and the supernatant decanted. The solids were resuspended in media and homogenized in a blender for 1 min. The solids were washed a second time as described above and the TSS (total suspended solids) measured. Sufficient inoculum was added to the media to obtain solids concentration of 10 mg/L. This mixture was adjusted to pH 7.0 and aerated overnight with CO2 free air.
- Pretreatment: None
- Concentration of sludge: 10 mg solids/L
- Initial cell/biomass concentration: Not reported
- Water filtered: Not applicable
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

act. ingr.

Initial conc.:

12.39 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 301 B guideline.
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): None used
- Test temperature: 22±1°C
- pH: 7
- pH adjusted: Not reported
- CEC (meq/100 g): Not applicable
- Aeration of dilution water: The test solutions (mineral media plus inoculum) were aerated overnight with CO2-free air before the addition of test substance or reference
- Suspended solids concentration: 10 mg solids/L
- Continuous darkness: Not reported
- Other: None

TEST SYSTEM
- Culturing apparatus: 1L bottle
- Number of culture flasks/concentration: Three
- Method used to create aerobic conditions: By aerating overnight with CO2 free air.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: Electrolytic respirometer (Coordinated Environmental Services Ltd., UK) was used for measuring CO2. The Soluble organic carbon (SOC) was determined using a Shimadzu Total Organic Carbon Analyzer.
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Details of trap for CO2: A conductivity probe immersed in 1% NaOH is used to measure the production of CO2. The base reservoirs for the conductivity probes were prepared one day prior to test initiation and allowed to equilibrate at 22° C. NaOH was degassed via filtration, precisely measured (18 mL) into the base cups and subsequently attached to the probes.
- Other:
- Test set up: Each sample assembly consisted of sample vessel with a conductivity probe, an electrolysis chamber (with cathode and anode electrodes in a saturated solution of CuSO4) and an empty bottle served as a reference vessel for the pressure transducer pressure transducer. All three components comprised the sensory assembly and were attached to the sample vessel.
- Test vessel setup: 20 mL of stock solution was added to each replicate, along with inoculum and media to reach a total volume of one liter in each test vessel.

SAMPLING
- Sampling frequency: Sampling was performed automatically via the integrated computer software and was programmed for a 12 h sampling interval following a 1 h stabilization period.
- Sampling method: Automated sampling
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not supplied
- Other: None

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, there were 3 replicates of the blank (no inoculum present).
- Abiotic sterile control: No
- Toxicity control: No
- Procedure control: Yes

STATISTICAL METHODS: Not reported

Reference substance:

benzoic acid, sodium salt

Remarks:

tested at 20 mg/L (12.12 mg Carbon/L); ThOC: 58.3%; TOC of its stock solution: 606.1 mg C/L.

Preliminary study:

Not applicable

Test performance:

No data

Parameter:

% degradation (CO2 evolution)

Value:

14.55

St. dev.:

3.12

Sampling time:

3 d

Remarks on result:

other: Mean value of 3 replicates, indicating start of 10 d window criterion

Parameter:

% degradation (CO2 evolution)

Value:

62.17

St. dev.:

2.77

Sampling time:

9 d

Remarks on result:

other: Mean value of 3 replicates, indicating that the 10 d window criterion was met

Parameter:

% degradation (CO2 evolution)

Value:

86.83

St. dev.:

4.87

Sampling time:

28 d

Remarks on result:

other: Mean value of 3 replicates, indicating final percentage of degradation

Details on results:

- Final SOC (in mg/L) was as found to be 0.3 and 0.4 for replicate 1 and 2 respectively. No data for replicate 3 was recorded due to instrument failure. The initial TOC was 12.39 mg/L.
- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’

Results with reference substance:

- The Reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window. Pass level of >60% reached after Day 5 in all replicates. (Mean values of all replicates on Day 1 = 17.4%, Day 5 = 61.6% and Day 28 = 93.6% TCO2)
For details on individual replicate results, please refer to 'Table 2' in the ‘Any other information on results incl. tables’

- Final mg Soluble Organic Carbon (SOC)/L were found to be 0.2, 0.2 and 0.3 mg/L in replicate 1, 2 and 3 respectively. The initial TOC was 12.12 mg/L.

Table 1. Ready biodegradation test of amine oxide 1214LP (SWH-338-025) (study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2
2	-	11.47
3	12.34	-
8	-	61.1
9	60.21	-
28	83.39	90.28

Note:No data was recorded for replicate 3 due to instrument failure

Table 2. Ready biodegradation test of reference substance(study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2	Replicate 3
1	16.73	18.65	16.73
5	62.60	63.39	61.34
28	92.52	96.74	91.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

C12/14 Amine oxide (Natural fatty alcohol) (C12/C14 AO) is readily biodegradable.

Executive summary:

A ready biodegradation test, following OECD 301B guideline, was conducted on C12/14 amine oxide (natural). The substance was tested at 20 mg active/L (12.39 mg TOC/L) and the inoculum was 10 mg activated sludge solids/L. The test treatment, inoculum blank and reference control were measured in triplicate. The deviation between replicate treatments was <20% (met the validity criteria of the guideline). 

Ten percent biodegradation of the test material was reached at Day 3 and 60% biodegradation was reached at Day 9 in all replicates. At Day 3, the average biodegradation was 14.6% (±3.12), and at Day 9, the average biodegradation was 62.2% (±2.77). Hence the test material met the 10 d window requirement for ready biodegradability.

The final level of biodegradation at 28 days was 86.8% (±4.87).

The reference material, sodium benzoate, reached 10% biodegradation at Day 1 and >60% after Day 4. It met the validity criteria established in the guideline for a reference material.

This ready biodegradation test is classified as acceptable, and satisfies the guideline requirements for the OECD 301B biodegradation screening test.

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 23, 2006 to September 20, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

However, this laboratory is in the process of attaining GLP status. This study was conducted in principle in accordance with GLPs.

Specific details on test material used for the study:

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

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from Fairfield waste water treatment plant (Fairfield, Ohio) on August 22, 2006. This WWTP receives primarily domestic wastewater.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: Sludge was aerated until use
- Storage length: Not reported
- Preparation of inoculum for exposure: The activated sludge solids were centrifuged for 20 minutes at 5000 rpm and the supernatant decanted. The solids were resuspended in media and homogenized in a blender for 1 min. The solids were washed a second time as described above and the TSS (total suspended solids) measured. Sufficient inoculum was added to the media to obtain solids concentration of 10 mg/L. This mixture was adjusted to pH 7.0 and aerated overnight with CO2 free air.
- Pretreatment: None
- Concentration of sludge: 10 mg solids/L
- Initial cell/biomass concentration: Not reported
- Water filtered: Not applicable
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

act. ingr.

Initial conc.:

14.75 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral media was made according to the OECD 301 B guideline.
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): None used
- Test temperature: 22±1°C
- pH: 7
- pH adjusted: Not reported
- CEC (meq/100 g): Not applicable
- Aeration of dilution water: The test solutions (mineral media plus inoculum) were aerated overnight with CO2-free air before the addition of test substance or reference
- Suspended solids concentration: 10 mg solids/L
- Continuous darkness: Not reported
- Other: None

TEST SYSTEM
- Culturing apparatus: 1L bottle
- Number of culture flasks/concentration: Three
- Method used to create aerobic conditions: Continuous aeration with CO2-free air.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: Electrolytic respirometer (Coordinated Environmental Services Ltd., UK) was used for measuring CO2. The Soluble organic carbon (SOC) was determined using a Shimadzu Total Organic Carbon Analyzer.
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Details of trap for CO2: A conductivity probe immersed in 1% NaOH is used to measure the production of CO2. The base reservoirs for the conductivity probes were prepared one day prior to test initiation and allowed to equilibrate at 22° C. NaOH was degassed via filtration, precisely measured (18 mL) into the base cups and subsequently attached to the probes.
- Other:
- Test set up: Each sample assembly consisted of sample vessel with a conductivity probe, an electrolysis chamber (with cathode and anode electrodes in a saturated solution of CuSO4) and an empty bottle served as a reference vessel for the pressure transducer pressure transducer. All three components comprised the sensory assembly and were attached to the sample vessel.
- Test vessel setup: 20 mL of stock solution was added to each replicate, along with inoculum and media to reach a total volume of one liter in each test vessel.

SAMPLING
- Sampling frequency: Sampling was performed automatically via the integrated computer software and was programmed for a 12 h sampling interval following a 1 h stabilization period.
- Sampling method: Automated sampling
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not supplied
- Other: None

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, there were 3 replicates of the blank (no inoculum present).
- Abiotic sterile control: No
- Toxicity control: No
- Procedure control: Yes

STATISTICAL METHODS: Not reported

Reference substance:

benzoic acid, sodium salt

Remarks:

tested at 20 mg/L (12.12 mg C/L); ThOC: 58.3%; TOC of its stock solution: 606.1 mg C/L

Preliminary study:

Not applicable

Test performance:

No data

Parameter:

% degradation (CO2 evolution)

Value:

14.3

St. dev.:

1.02

Sampling time:

3 d

Remarks on result:

other: Mean value of 3 replicates, indicating start of 10 d window criterion

Parameter:

% degradation (CO2 evolution)

Value:

64.51

St. dev.:

3.2

Sampling time:

10 d

Remarks on result:

other: Mean value of 3 replicates, indicating that the 10 d window criterion was met

Parameter:

% degradation (CO2 evolution)

Value:

88.93

St. dev.:

4.52

Sampling time:

28 d

Remarks on result:

other: Mean value of 3 replicates, indicating final percentage of degradation

Details on results:

- Final SOC in (mg/L) was found to be 0.4, 0.3 and 0.3 for replicate 1, 2 and 3 respectively. The initial TOC was 14.75 mg/L.
- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’

Results with reference substance:

- The Reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window. Pass level of >60% reached after Day 5 in all replicates. (Mean value of all replicates on Day 1 = 17.4%; Day 5 = 61.6% Day 28 = 93.6% TCO2)
For details on individual replicate results, please refer to 'Table 2' in the ‘Any other information on results incl. tables’

- Final mg Soluble Organic Carbon (SOC)/L were found to be 0.2, 0.2 and 0.3 mg/L in replicate 1, 2 and 3 respectively. The initial TOC was 12.12 mg/L.

Table 1. Ready biodegradation test of amine oxide 1214LP (SWF-866-097) (study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2	Replicate 3
2	-	-	10.43
3	13.54	13.89
8	-	-	61.75
9	60.51	-	-
10	-	61.51	-
28	92.07	83.75	90.98

 

Table 2. Ready biodegradation test of reference substance(study # 45598)

Time (days)	Percent TCO2
	Replicate 1	Replicate 2	Replicate 3
1	16.73	18.65	16.73
5	62.60	63.39	61.34
28	92.52	96.74	91.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

C12/14 Amine oxide (Synthetic alpha-olefin) (C12/14 AO) is readily biodegradable.

Executive summary:

A ready biodegradation test, following OECD 301B guideline, was conducted on C12/14 amine oxide (synthetic). The test substance was tested at 20 mg active/L (14.75 mg TOC/L)and the inoculum was 10 mg activated sludge solids/L. The test treatment, inoculum blank and reference control were measured in triplicate. The deviation between replicate treatments was <20% (met the validity criteria of the guideline). 

Ten percent biodegradation of the test material was reached at Day 3 and 60% biodegradation was reached at Day 10 in all replicates. At Day 3, the average biodegradation was 14.3% (±1.02), and at Day 10 the average biodegradation was 64.5% (±3.20). Hence the test material met the 10 d window requirement for ready biodegradability.

The final level of biodegradation at 28 days was 88.9% (±4.52).

The reference material, sodium benzoate, reached 10% biodegradation at Day 1 and >60% after Day 4. It met the validity criteria established in the guideline for a reference material.

This ready biodegradation test is classified as acceptable, and satisfies the guideline requirements for the OECD 301B biodegradation screening test.

Description of key information

Two studies are available for C10 AO showing that it is readily biodegradable meeting the 10-day window. These findings are supported by the results of studies performed using C12-14 AO.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In the key study [Noack M (1997)] the ready biodegradability of C10 AO was assessed according to OECD TG 301E under GLP. 97% degradation (based on DOC removal) was observed after 28 days when the innoculum (domestic sewage sludge) was exposed to the substance at a concentration of approximately 45.9 mg AO/L. The test was performed in duplicate. The substance reached 18 % degradation on Day 5 and 72% degradation on Day 7, hence meeting the 10-day window requirement for ready biodegradability.The reference substance, sodium acetate, reached 44 % degradation on Day 1 and 93 % by Day 5, meeting the validity criteria established in the guideline for a reference substance.

In a supporting study [Casteel K (2007)] the C10 AO was tested following OECD TG 301B at 14 mg C/L and an inoculum of activated sewage sludge solids. 13.96 % biodegradation of the test material was reached at Day 3 and 61.67 % biodegradation was reached at Day 9 (mean of 3 replicates). Hence the test material met the 10 d window requirement for ready biodegradability. The final level of biodegradation at 28 days was 91.22% (mean of 3 replicates). The reference substance, sodium benzoate, reached 17.4 % degradation on Day 1 and 61.6 % degradation on Day 5, meeting the validity criteria.

Several studies where C12 -14 AO was tested to OECD TG 301 B [Clarke (2005] [Casteel (2007)] [Hirschen (2001)], OECD TG 301 D [Balk & Hantink-De Rooij (1987)] or DIN 38 412 [Klein (1986)] are available. The substance was found to be readily biodegradable in all of these studies.

Based on these studies it is concluded that C10 AO is readily biodegradable.