Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

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

Study period:

23/04/2004 to 26/11/2004

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:

test item, reference substance, nutrient stock solutions and inoculum were applied individually into the test vessels

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

yes

Remarks:

test item, reference substance, nutrient stock solutions and inoculum were applied individually into the test vessels

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): the effluent of the municipal activated sludge plant of Pforzheim (Germany) was used as inoculum (not further specified)
- Preparation of inoculum for exposure: The inoculum was collected 2 days prior to the test. It was filtered through a coarse filter (producer Macherey & Nagel), the first 200 mL being discarded, and was shaken for one hour for starvation. The inoculum was kept under aerobic conditions in the period between sampling and application (ca. 48 h; sludge age at test iniatiation: ca. 48 h).
- Pretreatment: not reported
- Initial cell/biomass concentration: 3.12 x 10E4 bacterial cells (average value) in each test vessel

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: KH2PO4: 8.5 mg/L, K2HPO4 x 3 H2O: 28.5 mg/L, Na2HPO4 x 2H2O: 33.4 mg/L, NH4Cl: 0.5 mg/L, MgSO4 x 7 H2O: 22.5 mg/L, CaCl2 x 2 H2O: 36.4 mg/L, and FeCl3 x 6 H2O: 0.25 mg/L. The medium corresponds to the composition reported in the guideline with the exception of K2HPO4 x 3 H2O: 28.5 mg/L 21.75 mg/L
- Additional substrate: not reported
- Solubilising agent (type and concentration if used): no (for water solubility see chapter 4.8, reference: GAB 2005)
- Test temperature: 20 +/- 2°C
- pH: not reported
- pH adjusted: no data
- Aeration of dilution water: yes (for at least 60 min to achieve oxygen saturation; thereafter the water was allowed to stand for 24 h without aeration at test temperature)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD flasks with ground-in-glass stoppers
- Number of culture flasks/concentration: 13 (3-fold test assay for measurement date: 7, 14, 21, and 28 d; on day 0 one culture flask was checked for oxygen content)
- Method used to create aerobic conditions: aeration of dilution water for at least 60 min to achieve oxygen saturation; thereafter the water was allowed to stand for 24 h without aeration at test temperature
- Measuring equipment: WTW Microprocessor Oximeter OXI 340 (oxygen measurement; calibrated electrode)
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: on day 0 and after 7, 14, 21, and 28 three bottles were removed and analysed for O2 content
- Sampling method: bottles were removed and analysed for O2 content
- Sample storage before analysis: not reported

CONTROL AND BLANK SYSTEM
- Inoculum blank: performed
- Toxicity control: performed
- Other: functional control with reference substance performed

STATISTICAL METHODS: % biodegradation=BOD x 100/COD (COD (chemical oxygen demand)= 2.09 mg O2/mg test item; for details see below)

Reference substance:

benzoic acid, sodium salt

Remarks:

2 mg/L

Test performance:

The ready biodegradability of the test substance was investigated in a study conducted according to OECD Guideline 301 D and EU-Method C.4-E.

Parameter:

% degradation (O2 consumption)

Value:

66.2

Sampling time:

28 d

Remarks on result:

other: ready biodegradable and fulfilling the 14-d window criterion

Details on results:

The test item partially unsaturated TEA-Esterquat was biodegraded by 66.2 after 28 d and can therefore regarded as readily biodegradable and fulfilling the 14-d window criterion. The reference substance benzoic acid, sodium salt was biodegraded by 76.6% after day 14 and thus fulfilling the requirements of the guideline. The degradation of the toxicity control was >25% after 14 days. Therefore toxic effects to the inoculum can be excluded according to guideline (for details see below).

Results with reference substance:

The reference substance benzoic acid, sodium salt (2 mg/L) was biodegraded by 76.6% after 14 d and thus fulfilling the requirements of the guideline (for details see below).

The ready biodegradability of partially unsaturated TEA-Esterquat was determined in a study conducted according to OECD Guideline 301 D and EU-Method C.4 -E. The following results were obtained

Degradation rates (in %) of reference substance, test item, and in the toxicity control

a. reference substance (2 mg/L)

Bottle	7 d	14 d	21 d	28
1	68.9	77.8	90.4	92.2
2	73.1	73.1	82.0	92.2
3	67.7	79.0	80.2	98.8
Mean value	69.9	76.6	84.2	94.4

b. Partially unsaturated TEA-Esterquat (2 mg/L)

Bottle	7 d	14 d	21 d	28
1	20.5	43.5	60.2	64.0
2	23.4	46.3	74.5	65.0
3	23.4	46.3	54.9	69.7
Mean value	22.5	45.4	63.2	66.2

c. Toxicity control (1 mg/L partially unsaturated TEA-Esterquat and 1 mg/L Na benzoate)

Bottle	7 d	14 d	21 d	28
1	41.5	56.3	63.2	80.8
2	52.1	57.9	69.1	91.4
3	54.7	63.8	73.9	90.3
Mean value	49.4	59.3	68.7	87.5

The test substance was readily biodegradable (66.2% biodegradation after 28 days) and fulfilling the 14-d window criterion (day 7 -21). The reference substance benzoic acid, sodium salt (2 mg/L) was biodegraded by 76.6% after 14 d and thus fulfilling the requirements of the guideline. The degradation of the toxicity control was > 25% after 14 days; therefore toxic effects to the inoculum can be excluded. The residual O2 content did not fall below 0.5 mg/L at any time. In the inoculum blank, O2 consumption was <1.5 mg/L O2 after 28 d (0.57 mg/L O2 on average).

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The ready biodegradability of partially unsaturated TEA-Esterquat was determined in a study conducted according to OECD Guideline 301 D and EU-Method C.4 -E. The test substance was readily biodegradable (66.2% biodegradation after 28 days) and fulfilling the 14-d window criterion (day 7 -21).The reference substance benzoic acid, sodium salt (2 mg/L) was biodegraded by 76.6% after 14 d and thus fulfilling the requirements of the guideline.The degradation of the toxicity control was > 25% after 14 days; therefore toxic effects to the inoculum can be excluded. The residual O2 content did not fall below 0.5 mg/L at any time. In the inoculum blank, O2 consumption was <1.5 mg/L O2 after 28 d (0.57 mg/L O2 on average).

Executive summary:

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 D and EU-Method C.4 -E over a period of 28 days and using domestic sewage as inoculum. The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 1 mg/L test item and 1 mg/L reference compound were performed.

This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation.

The test item proved to be readily biodegradable and fulfilling the 14-d window criterion. The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item >25% biodegradation based on oxygen consumption occurred within 14 d thus indicating that the test item was not inhibitory at the concentration tested.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-09-15 to 2005-10-14

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)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was collected from the sewage treatment plant at Hildesheim (Germany) which treats mostly municipal sewage (industrial waste: 30-33%, referring to BOD).
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water and maintained in an aerobic condition by aeration for 4 h and then homogenized with a mixer. Thereafter the sludge was filtered with folded filter and aerated with CO2-free air for 5 d.
- Pretreatment: no
- Initial cell/biomass concentration: 10E7-10E8 CFU/L

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral nutrient solution acc. to OECD 301 B/CO2 Evolution Test
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 22 +/- 2°C
- pH: initial pH: not reported; at test termination: 7.44-7.46 (test vessel with test substance)
- pH adjusted: not reported
- Aeration of dilution water: not reported
- Continuous darkness: testing was conducted in brown glass bottles

TEST SYSTEM
- Culturing apparatus: 5-L brown glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The test vessels were aerated for 24 h with CO2-free air.
- Measuring equipment: pH-Meter, Multilab 340i, WTW; Thermohygrograph, type 3.015/3 K, fabr.-no. 9003146; Flow meter, KROHNE DUISBURG TYP DK 800 PV; Burette, BÜRKLE; Dispenser ROTH; Magnetic stirrer, VARIOMAG Mono; Membrane pump, BEHR
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: 3 gas wash bottles were connected in series to the test vessels containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

SAMPLING
- Sampling frequency: sampling was performed on day 1, 4, 6, 8, 11, 14, 19, 21, 25, 28, and 29 (on day 28 1 mL of 37% HCl was added to each of the test vessels)

CONTROL AND BLANK SYSTEM
- Inoculum blank: performed
- Abiotic sterile control: not reported
- Toxicity control: performed
- Other: functional/procedural control (reference substance) performed

Reference substance:

acetic acid, sodium salt

Remarks:

(35 mg/L)

Parameter:

% degradation (CO2 evolution)

Value:

> 10

Sampling time:

4 d

Parameter:

% degradation (CO2 evolution)

Value:

> 60

Sampling time:

15 d

Parameter:

% degradation (CO2 evolution)

Value:

75

Sampling time:

28 d

Results with reference substance:

The reference substance sodium acetate was biodegraded by 75% after 14 d and thus fulfilling the requirements of the guideline (>60% biodegradation after 14 d).

The test substance proved to be readily biodegradable (75 % biodegradation on average after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992). The reference substance sodium acetate was biodegraded by 75% after 14 d and thus fulfilling the requirements of the guideline (>60% biodegradation after 14 d). In the toxicity control containg 35 mg/L sodium acetate and 20 mg/L test substance a biodegradation of 64% occurred within 14 d. The results indicate that the test substance was not inhibitory to the microorganisms and the requirements of the guideline (>=25% biodegradation) are fulfilled. The difference of extremes of replicate values of removal of the test substance at the end of the test was <20%. The total CO2 evolution in the control at the end of the test was 44.4 mg/L and is slightly higher than reported in the guideline (should normally <=40 mg/L and not exceed 70 mg/L).

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance proved to be readily biodegradable (75 % biodegradation on average after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992).

Executive summary:

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992) over a period of 28 days and using municipal activated sludge as inoculum. The biodegradation rate was determined by measurement of CO2 evolution. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L test substance and 35 mg/L reference compound were performed. The test substance proved to be readily biodegradable (75 % biodegradation on average after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992). The reference substance sodium acetate was biodegraded by 75% after 14 d and thus fulfilling the requirements of the guideline (>60% biodegradation after 14 d). In the toxicity control containg 35 mg/L sodium acetate and 20 mg/L test substance a biodegradation of 64% occurred within 14 d. The results indicate that the test substance was not inhibitory to the microorganisms and the requirements of the guideline (>=25% biodegradation) are fulfilled. The difference of extremes of replicate values of removal of the test substance at the end of the test was <20%. The total CO2 evolution in the control at the end of the test was 44.4 mg/L and is slightly higher than reported in the guideline (should normally <=40 mg/L and not exceed 70 mg/L). This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.06.2005 to 01.07.2005

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)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht (Germany) was used as inoculum. The wwtp clarifies predominantly domestic wastewater. The inoculum was sampled on May 31st, 2005.
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in aerated tap water.
- Pretreatment: no
- Concentration of sludge: 30 mg/L dry solids

Duration of test (contact time):

28 d

Initial conc.:

ca. 20 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 21.0-22.5°C
- pH: not reported
- pH adjusted: not reported
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L dry solids
- Continuous darkness: diffuse light

TEST SYSTEM
- Culturing apparatus: 2000 mL gas wash bottles were used as reactor
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The sealed test vessels containing activated sludge and mineral medium were aerated with CO2-free air at a rate of 50-100 mL/min overnight.
- Measuring equipment: TOC-5000A, Shimadzu Deutschland
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: Two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH.

SAMPLING
- Sampling frequency: sampling was performed on day 0 and after 4, 7, 11, 14, 21, and 28 (on day 28 1 mL concentrated HCl was added to the reactor vessels to release the CO2 dissolved in water)

CONTROL AND BLANK SYSTEM
- Inoculum blank: performed
- Abiotic sterile control: not performed
- Toxicity control: performed
- Other: functional/procedural control (reference substance) performed

Reference substance:

benzoic acid, sodium salt

Remarks:

(20 mg/L TOC)

Parameter:

% degradation (CO2 evolution)

Value:

78.8

Sampling time:

7 d

Parameter:

% degradation (CO2 evolution)

Value:

98.9

Sampling time:

28 d

Results with reference substance:

The reference substance sodium benzoate was biodegraded by 95.5% after 14 d.

The ready biodegradability of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test). The test item proved to be readily biodegradable (78.8% biodegradation after 7 d; 98.9% biodegradation after 28 d). The reference substance sodium benzoate was biodegraded by 95.5% after 14 d. The degradation in the toxicity control (ca. 20 mg/L TOC reference and test substance each) was 96.8% after 14 d and 98.6% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 -evolution of the blank flasks was 28.5 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10 -d window and at the end of the test was <20%.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Partially unsaturated TEA-Esterquat proved to be readily biodegradable (78.8% biodegradation after 7 d; 98.9% biodegradation after 28 d) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test).

Executive summary:

The ready biodegradation of the partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test over a period of 28 days and using municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using ca. 20 mg/L TOC reference and test substance each were performed. The test item proved to be readily biodegradable (78.8% biodegradation after 7 d; 98.9% biodegradation after 28 d). The reference substance sodium benzoate was biodegraded by 95.5% after 14 d. The degradation in the toxicity control (ca. 20 mg/L TOC reference and test substance each) was 96.8% after 14 d and 98.6% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 -evolution of the blank flasks was 28.5 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10 -d window and at the end of the test was <20%. This study is regarded as reliable without restriction and satisfies the guideline requirements for ready/inherentbiodegradation.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

ECETOC Anaerobic Biodegradation (Technical Report No. 28)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

anaerobic

Inoculum or test system:

other: anaerobic sludge bacteria

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water PlC, sewage Works,Raynesway, Derby, Derbyshire

Duration of test (contact time):

ca. 56 d

Initial conc.:

ca. 77.51 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: dissolved inorganic carbon (DIC)

Parameter followed for biodegradation estimation:

other: gas production

Details on study design:

TEST CONDITIONS
- Composition of medium: KH2PO4 (0.27 g/L); Na2HPO4 x 12 H2O (1.12 g/L); NH4Cl (0.53 g/L); CaCl2 x 2H2O (0.075 g/L); MgCl2 X 6H2O (0.10 g/L); FeCl2 X 4H2O (0.001 mg/L)
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 35°C
- pH: 7.2 at start and 6.9 at the end of the test
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water: no
- Suspended solids concentration: 3.66 g/L
- Continuous darkness: yes



TEST SYSTEM
- Culturing apparatus: 125 mL Wheaton bottles (total volume 160 L with air tight seals and Teflon septa. culture vessels were housed in a water bath at 35°C
- Number of culture flasks/concentration: test concentration 4 replicates; reference substance sodium benzoate 4 replicates; controls (culture medium) 8 replicates
- Method used to create anaerobic conditions: culture medium was purged with oxygen free nitrogen before the test; Resazurin was added at a concentration of 1 mg/L to all test, standard and control vessels to confirm anaerobic conditions.
- Measuring equipment: Dohrmann DC190 Carbon Analyser; Watson-Smith hand held precision pressure meter;
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used:



SAMPLING
- Sampling frequency: dissolved inorganic carbon (DIC) was sampled on days 0 and 56; pH was measured at start and at the end of the test; Gas was measured at days 0, 1, 7, 14, 21, 28, 35, 42, 49, 56
- Sampling method: dissolved inorganic carbon (DIC) was determined using a Dohrmann DC190 Carbon Analyser; gas measurements were performed by pressure measurement with a Watson-Smith hand held precision pressure meter attached to a miniature three-way inert valve and syringe needle. Atmospheric pressure was recorded daily


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

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (inorg. C analysis)

Value:

76

Sampling time:

56 d

Remarks on result:

other: dissolved inorgan ic carbon (DIC)

Parameter:

other: gas evolution

Value:

72

Sampling time:

56 d

Table 1: Percentage Biodegradation for Sodium Benzoate and partially unsaturated TEA-Esterquat

(Calculated from volume of gas produced)

Day	Sodium benzoate % degradation	Partially unsaturated TEA-Esterquat % degradation
1	0	22
7	40	54
14	74	59
21	71	64
28	70	64
35	69	64
42	73	71
49	74	72
56	74	72

 

Table 2: Sodium Benzoate and Partially unsaturated TEA-Esterquat Mean Values and Standard Deviations of Dissolved Inorganic Carbon Measurements

Day	Mean DIC produced (mg/L)
	Control		Sodium benzoate		Partially unsaturated TEA-Esterquat
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0	6.23	0.38	7.10	0.54	7.56	0.69
56	30.73	2.89	39.27	2.00	33.88	1.42

 

Based on DIC measurements degradation of partially unsaturated TEA-Esterquat was calculated for 76 %.

Sodium benzoate attained 89 % degradation based on the DIC measurements.

Validity criteria fulfilled:

yes

Interpretation of results:

other: biodegradable under anaerobic conditions

Conclusions:

The test material attained 76% degradation after 56 days and therefore can be considered as biodegradable under anaerobic conditions.

Executive summary:

The anaerobic biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to ECETOC Anaerobic Biodegradation (Technical Report No. 28) over a period of 56 days and using anaerobic sludge bacteria from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of dissolved inorganic carbon (DIC). This study is regarded as reliable with restriction and satisfies the guideline requirements for anaerobic biodegradation. The test material attained 76% degradation after 56 days and therefore can be considered as biodegradable under anaerobic conditions.

Reference 5

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 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: sewage sludge micro-organisms

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at belper, Derbyshire, UK

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 17.1 mg/L

Based on:

test mat.

Initial conc.:

ca. 10 other: mg C/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: according to OECD 301 B
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 21°C
- pH: no data
- CEC (meq/100 g): no data
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes



TEST SYSTEM
- Culturing apparatus: sealed culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration prior the test
- Measuring equipment: Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser.
- Test performed in closed vessels due to significant volatility of test substance:
- Details of trap for CO2 and volatile organics if used: CO2 absorber vessel


SAMPLING
- Sampling frequency: sampling on day 0 ,1 ,2 ,3 ,6 ,8 ,14 ,16 ,20 ,22 ,24 ,27, 28 and 29 (first absorber vessel); sampling on day 0 and day 29 (second absorber vessel)
- Sampling method: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water. The samples from the first absorber vessel were analysed for CO2 using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. Samples (40 and 50 micro L were injected into the IC (Inorganic Carbon Channel) of the TOC analyser. On day 0 and 28 samples (20 mL) were removed from the culture vessels and centrifuged (3500 rpm, 15 min) prior to Dissolved Organic Carbon (DOC) analysis. The samples were analysed for DOC using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. Samples ( 40 or 50 microL) were injected into the TC (Total Carbon) and IC (Inorganic Carbon) channels of the analyser. Each analysis was carried out in triplicate.
- Sample storage before analysis: samples were analysed immediately, exeption: sample from day 12 was stored at -20°C, however this sample was not analysed for CO2, as previous and subsequent samples showed that the level of degradation did not increase significant



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

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

78

Sampling time:

28 d

Table 1: Percentage Biodegradation values

Day	% Degradation Sodium benzoate	% Degradation Partially saturated TEA-EQ	% Degradation   Partially saturated TEA-Esterquat plus Sodium benzoate Toxicity control
1	15	5	11
2	35	9	20
3	53	15	24
6	73	29	42
8	78	52	45
12	78	54	40
14	86	63	39
16	82	69	42
20	85	72	44
22	86	68	47
24	88	71	49
27	91	78	51
28	94	78	57
29*	89	74	56

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

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 78% degradation after 28 days and therefore can be considered as readily biodegradable.

Executive summary:

The ready biodegradation of partially saturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) over a period of 28 days and using activated sludge from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2 evolution. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L test item and 10 mg/L reference compound were performed. This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation. The test item proved to be readily biodegradable and fulfilling the 10-d window criterion. The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item 39% biodegradation based on ThCO2 occurred within 14 d thus indicating that the test item was not inhibitory at the concentration tested.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.08.2005 to 21.09.2005

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)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht (Germany) was used as inoculum (3.8 g/L dry solids). The wwtp clarifies predominantly domestic wastewater. The inoculum was sampled on August 16th, 2005.
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in aerated tap water.
- Pretreatment: no
- Concentration of sludge: 30 mg/L dry solids

Duration of test (contact time):

28 d

Initial conc.:

20.1 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 20-22°C
- pH: not reported
- pH adjusted: not reported
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L dry solids
- Continuous darkness: diffuse light

TEST SYSTEM
- Culturing apparatus: 2000 mL gas wash bottles were used as reactor
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The sealed test vessels containing activated sludge and mineral medium were aerated with CO2-free air at a rate of 50-100 mL/min overnight.
- Measuring equipment: TOC-5000A, Shimadzu Deutschland
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: Two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH.

SAMPLING
- Sampling frequency: sampling was performed on day 3, 6, 10, 14, 21, and 28 (on day 28 1 mL concentrated HCl was added to the reactor vessels to release the CO2 dissolved in water)

CONTROL AND BLANK SYSTEM
- Inoculum blank: performed
- Abiotic sterile control: not performed
- Toxicity control: performed
- Other: functional/procedural control (reference substance) performed

Reference substance:

benzoic acid, sodium salt

Remarks:

(20 mg/L organic carbon)

Parameter:

% degradation (CO2 evolution)

Value:

25.2 - 28.9

Sampling time:

3 d

Parameter:

% degradation (CO2 evolution)

Value:

58 - 58.3

Sampling time:

14 d

Parameter:

% degradation (CO2 evolution)

Value:

71

Sampling time:

28 d

Results with reference substance:

The reference substance was biodegraded by 74% (mean of 3 measurements) after 14 d.

The ready biodegradability of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test). The test item proved to be readily biodegradable (71.4% biodegradation after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals). The reference substance was biodegraded by 74% after 14 d. The degradation in the toxicity control (20 mg/L TOC reference and test substance each) was 75.2% after 14 d and 78.3% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 -evolution of the blank flasks was 42.8 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10 -d window and at the end of the test was <20%.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Partially unsaturated TEA-EQ proved to be readily biodegradable (71.4% biodegradation after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test).

Executive summary:

The ready biodegradability of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) over a period of 28 days and using activated sludge from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2. The test item proved to be readily biodegradable (71.4% biodegradation after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals). The reference substance was biodegraded by 74% after 14 d. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L TOC reference and test substance each were performed. The reference substance was biodegraded by 74% after 14 d. The degradation in the toxicity control (20 mg/L TOC reference and test substance each) was 75.2% after 14 d and 78.3% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 -evolution of the blank flasks was 42.8 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10 -d window and at the end of the test was <20%. This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation.

Description of key information

readily biodegradable under aerobic conditions (OECD TG 301 D (Closed Bottle Test) / OECD TG 301 B (CO2 Evolution Test)); GLP; RL1
readily biodegradable under anaerobic conditions (ECETOC Anaerobic Biodegradation (Technical Report No. 28)); GLP; RL2

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

 

Summary

One test according to OECD Guideline 301 D (Closed Bottle Test) and four tests according to OECD Guideline 301 B (CO2 Evolution Test) proved the ready biodegradation of partially unsaturated TEA-Esterquat under aerobic conditions.

One study according to ECETOC Anaerobic Biodegradation (Technical Report No. 28) proved the biodegradation of partially unsaturated TEA-Esterquat under anaerobic conditions.

 

Studies in detail

Aerobic biodegradation

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 D and EU-Method C.4 -E over a period of 28 days and using domestic sewage as inoculum. The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 1 mg/L test item and 1 mg/L reference compound were performed. This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation. The test item proved to be readily biodegradable and fulfilling the 14-d window criterion. The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item >25% biodegradation based on oxygen consumption occurred within 14 d thus indicating that the test item was not inhibitory at the concentration tested.

 

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test over a period of 28 days and using municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using ca. 20 mg/L TOC reference and test substance each were performed. The test item proved to be readily biodegradable (78.8% biodegradation after 7 d; 98.9% biodegradation after 28 d). The reference substance sodium benzoate was biodegraded by 95.5% after 14 d. The degradation in the toxicity control (ca. 20 mg/L TOC reference and test substance each) was 96.8% after 14 d and 98.6% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 evolution of the blank flasks was 28.5 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10-d window and at the end of the test was <20%.

 

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) over a period of 28 days and using activated sludge from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2 evolution. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L test item and 10 mg/L reference compound were performed. This study is regarded as reliable without restriction and satisfies the guideline requirements for ready biodegradation. The test item proved to be readily biodegradable and fulfilling the 10-d window criterion. The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item 39% biodegradation based on ThCO2 occurred within 14 d thus indicating that the test item was not inhibitory at the concentration tested.

 

The ready biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992) over a period of 28 days and using municipal activated sludge as inoculum. The biodegradation rate was determined by measurement of CO2 evolution. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L test substance and 35 mg/L reference compound were performed. The test substance proved to be readily biodegradable (75 % biodegradation on average after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals) in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test; 1992). The reference substance sodium acetate was biodegraded by 75% after 14 d and thus fulfilling the requirements of the guideline (>60% biodegradation after 14 d). In the toxicity control containg 35 mg/L sodium acetate and 20 mg/L test substance a biodegradation of 64% occurred within 14 d. The results indicate that the test substance was not inhibitory to the microorganisms and the requirements of the guideline (>=25% biodegradation) are fulfilled. The difference of extremes of replicate values of removal of the test substance at the end of the test was <20%. The total CO2 evolution in the control at the end of the test was 44.4 mg/L and is slightly higher than reported in the guideline (should normally <=40 mg/L and not exceed 70 mg/L).

 

The ready biodegradability of partially unsaturated TEA-Esterquat was investigated in a study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) over a period of 28 days and using activated sludge from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of CO2. The test item proved to be readily biodegradable (71.4% biodegradation after 28 d; according to the revised OECD Guidelines the 10-d window should not be applied to interpret the results of a test with a mixture of structurally similar chemicals). The reference substance was biodegraded by 74% after 14 d. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 20 mg/L TOC reference and test substance each were performed. The reference substance was biodegraded by 74% after 14 d. The degradation in the toxicity control (20 mg/L TOC reference and test substance each) was 75.2% after 14 d and 78.3% after 28 d. Thus no inhibition of the inoculum was caused by the test substance. The highest mean CO2 -evolution of the blank flasks was 42.8 mg/L (according to guideline this value should not exceed 40 mg/L and must be below 70 mg/L). The IC content in the test vessel was <5% of the TOC introduced with the test substance. The difference of extremes of replicate values at the end of the 10 -d window and at the end of the test was <20%.

 

Anaerobic biodegradation

The anaerobic biodegradation of partially unsaturated TEA-Esterquat was investigated in a study conducted according to ECETOC Anaerobic Biodegradation (Technical Report No. 28) over a period of 56 days and using anaerobic sludge bacteria from a municipal wastewater treatment plant as inoculum. The biodegradation rate was determined by measurement of dissolved inorganic carbon (DIC). This study is regarded as reliable with restriction and satisfies the guideline requirements for anaerobic biodegradation. The test material attained 76% degradation after 56 days and therefore can be considered as biodegradable under anaerobic conditions.