Reaction product of 2-(2-aminoethoxy)ethanol and 2,2',2''-nitrilotriethanol and heptanoic acid and isononanoic acid

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-Feb-2006 to 31-Mar-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

in the version dated 17 July 1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-nitrilotriethanol

Constituent 2

Reference substance name:

2-(2-aminoethoxy)ethanol

EC Number:

213-195-4

EC Name:

2-(2-aminoethoxy)ethanol

Cas Number:

929-06-6

Molecular formula:

C4H11NO2

IUPAC Name:

2-(2-aminoethoxy)ethanol

Constituent 3

Reference substance name:

Heptanoic acid

EC Number:

203-838-7

EC Name:

Heptanoic acid

Cas Number:

111-14-8

Molecular formula:

C7H14O2

IUPAC Name:

heptanoic acid

Constituent 4

Reference substance name:

Isooctanoic acid

EC Number:

246-617-0

EC Name:

Isooctanoic acid

Cas Number:

25103-52-0

Molecular formula:

C8H16O2

IUPAC Name:

isooctanoic acid

Constituent 5

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Study design

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test System (Inoculum): Prior to use a sample of activated sludge from the sewage plant at Taunusstein-Bleidenstadt was washed twice with mineral nutrient solution of the CO2 Evolution Test to eliminate organic components and carbonates from the sludge. After resolution with mineral nutrient medium the sludge was aerated by means of compressed humidified air for about four hours. Before use as inoculum for the CO2-Evolution-Test the sludge was homogenised in a "Waring Blender" at low speed for 2 minutes and then filtered through a cotton filter previously carefully rinsed with deionised water. The filtrate was used as inoculum (1% of the final volume of the test solution) on the same day of preparation.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 22.46 mg/L

Based on:

test mat.

Remarks:

1st Test Solution: 78.6 mg/3.5 L.

Initial test substance concentration 2

Initial conc.:

ca. 22.57 mg/L

Based on:

test mat.

Remarks:

2nd Test Solution: 79.0 mg/3.5 L.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Application Method:
According to the carbon content of the test item the amount of test item to be applied was calculated to give a final concentration of 10 to 20 mg TOC/L (Total Organic Carbon). For preparation of the test solutions the follo¬wing stock solutions of mineral nutrient salts were prepared:

- 8.50 g KH2PO4
21.75 g K2HPO4
33.40 g Na2HPO4 · 2 H2O
0.50 g NH4Cl
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at + 4°C).

- 36.40 g CaCl2 · 2 H2O (≙ 27.5 g CaCl2)
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at + 4°C).

- 22.50 g MgSO4 · 7 H2O
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at + 4°C).

- 0.125 g FeCl3 · 6 H2O
were diluted in 0.5 L Ultrapure Water [Seral, Purelab plus] (this solution was prepared freshly before use in the test series).

The test solutions contained in a total volume of 3500 mL: 35 mL of the inoculum, 3.5 mL of the ferrichloride solution, 3.5 mL of the magnesium sulfate solution, 3.5 mL of the calcium chloride solution, and 35 mL of the phosphate mixture solution. The test item was given directly into the test solutions. At time t0 78.6 mg of the test item were given into the first test solution and 79.0 mg were given into the second test solution. The final (calculated) concentration in the test solutions was about 10 mg C/L.
For Blank correction two test solutions were prepared in the same way as the test solutions with the test item, but without the addition of any test- or control item. At the same time one test solution containing 120.9 mg sodiumbenzoate/3.5 L (≈ 20 mg C/L) of test solution was run in parallel.
One further test solution was prepared using 120.9 mg sodiumbenzoate and 78.2 mg of the test item for toxicity control.
Before starting of the test the mineral nutrient solutions with inoculation but without test item were aerated with CO2 - free air for 24 h in order to purge the system off CO2. Within this study the test solutions were stirred by means of magnetic stirrers in order to distribute the test item (or control item) and oxygen in a way to dissolve them at the maximum solubility.


Dosing, Frequency and Duration of Application: The test item was introduced into the test solution in one step.


Description of the Course of Study:
≈5-litre carboys covered with aluminium foil served as test vessels. They were closed with stoppers with tubing for gas inlet and outlet (gas exit line). Before starting the test, the mineral nutrient solution with inoculum but without any test component were aerated for 24 hours with CO2-free compressed air (use of a 'CO2 scrubbing apparatus' described in the OECD Test Guideline 301 B), in order to purge the system of carbon dioxide. Hereafter the test was started by addition of the test item. The gas outlet (exit air line) was connected to three CO2 absorber bottles filled with 100 mL 0.025 N Ba(OH)2 (connection in series), and bubbling of CO2 free compressed air through the solution was continued. Periodically (if necessary) the CO2 absorber nearest the test vessel was removed for analysis, and a new CO2 absorber was connected farest the carboy. Precipitation of BaCO3 in the second CO2 trap indicated that the absorber bottle nearest the test vessel had to be changed and analysed for CO2.
Titrations were performed at t2d, t6d, t8d, t12d, t15d, t20d, t28d, and t29d.


Temperature: Temperature was measured daily and documented by the electronic registration system HAMSTER®. Values within the biologically active phase of the test were in the range of 19.5 to 20.6 °C (mean 20.1°C).

Aeration of the Test Solutions: The aeration rate of the test system was controlled at 4 L/h being assured by precise flowmeters (Fisher & Porter, Göttingen, FRG).

Reference substance

Reference substance:

other: A reference item was not used.

Remarks:

Control Item: Sodiumbenzoate.

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

mean degradation value

Value:

82

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Remarks:

1st Test Solution, Test Item

Value:

92

Sampling time:

28 d

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Remarks:

2nd Test Solution, Test Item

Value:

72

Sampling time:

28 d

Details on results:

Calculated from the organic carbon content of the test item and the measured CO2 generation, 92% of the theoretical CO2 (ThCO2) has been generated by the test item within 28d of test period in the first culture with 78.6 mg of test item. 72 % of the theoretical CO2 (ThCO2) has been generated by the test item within 28d in the second culture with 79.0 mg of test item. The mean degradation value of the test item was 82%. The “10-days-window” was met within both test solutions. Thus, the test item may be regarded as “readily biodegradable". The control item sodium benzoate was degraded 90%, the "10-days-window" being met within 6 days (70%). The total CO2-evolution of the Blank was 71.7 mg CO2. Thus the test is regarded to be valid. The results obtained with the toxicity control indicate that there was no toxicity of the test item towards microorganisms at the concentration used within the test.

Any other information on results incl. tables

Results of Testing Biodegradability according to OECD 301 B – 1st Test Solution, Test Item

Concentration: 78.6 mg/3.5 L / ≈10 mg TOC/L. ThCO2: 1624.82 mg CO2/g test item.

Theoretical amount of CO2 being generated if the test item is completely transformed to CO2: 127.71 mg CO2/ 3500 mL test solution.

Date	Time [d]	1st Test Solution with ≈10 mg C/L mg CO2 generated in the test solution, cumulative	% TCO2 (= % Biodegradation)
02-10-06	2	4.72	4
02-14-06	6	20.35	16
02-16-06	8	42.26	33
02-20-06	12	64.67	51
02-23-06	15	78.92	62
02-28-06	20	87.63	69
03-08-06	28	107.00	84
03-09-06	29	117.25	92

 

Results of Testing Biodegradability according to OECD 301 B  – 2nd Test Solution, Test Item

Concentration: 79.0 mg/3.5 L / ≈10 mg TOC/L. ThCO2: 1624.82 mg CO2/g test item.

Theoretical amount of CO2 being generated if the test item is completely transformed to CO2: 128.36 mg CO2/ 3500 mL test solution.

Date	Time [d]	1st Test Solution with ≈10 mg C/L mg CO2 generated in the test solution, cumulative	% TCO2 (= % Biodegradation)
02-10 -06	2	2.44	2
02-14-06	6	13.26	10
02-16-06	8	23.16	18
02-20-06	12	44.96	35
02-23-06	15	59.04	46
02-28-06	20	69.25	54
03-08 -06	28	86.82	68
03-09-06	29	91.90	72

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item is readily biodegradable. (OECD Guideline 301B(28d): 82%, the “10-days-window” was met).

Executive summary:

Test Item was tested for biodegradability according to 'CO2-Evolution-Test' (OECD Guideline 301B). Calculated from the organic carbon content of the test item and the measured CO2 generation, 92% of the theoretical CO2 (ThCO2) has been generated by the test item within 28d of test period in the first culture with 78.6 mg of test item. 72 % of the theoretical CO2 (ThCO2) has been generated by the test item within 28d in the second culture with 79.0 mg of test item. The mean degradation value of the test item was 82%. The “10-days-window” was met within both test solutions. Thus, the test item may be regarded as “readily biodegra¬dable". The control item sodium benzoate was degraded 90%, the "10-days-window" being met within 6 days (70%). The total CO2-evolution of the Blank was 71.7 mg CO2. Thus the test is regarded to be valid. The results obtained with the toxicity control indicate that there was no toxicity of the test item towards microorganisms at the concentration used within the test.