N,N-bis(2,3-epoxypropyl)aniline

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2013 - 02 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ISO 9888

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

EC Number:

275-662-9

EC Name:

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

Cas Number:

71604-74-5

Molecular formula:

C15H19NO4

IUPAC Name:

3-(oxiran-2-ylmethoxy)-N,N-bis(oxiran-2-ylmethyl)aniline

Test material form:

liquid: viscous

Details on test material:

- Description : Clear slightly yellow very viscous liquid
- Test substance storage : In refrigerator (2-8°C) in the dark
- Stability under storage conditions : Stable
- Molecular formula : C15H19NO4
- Molecular weight : 277.3
- CAS Number : 71604-74-5

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source : The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.

- Preparation of the sludge : The sludge was coarsely sieved and washed with mineral medium. The batch of sludge was used within 6 hours of sampling and aerated until required. A small amount of the sludge was weighed and dried at ca. 105°C. From this result the amount of suspended solids (SS) was calculated and adjusted to 3.0 g/l.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

224.3 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.
- Test temperature: between 20 and 22°C.
- pH: between 6.5 and 7.9, except for the reference substance on day 7, 10 and 17. On day 7 the pH was 8.2 (adjusted to 7.5), on day 10 the pH was 8.2 (adjusted to 7.3) and on day 17 the pH was 6.3 (adjusted to 7.1).
- Oxygen concentrations: between 7.4 and 9.2 mg/l.
- Aeration of dilution water: the test is aerated continuoisly.
- Suspended solids concentration in the test mixtures: 0.75 g SS/l.
- The ratio of inoculum and m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline or reference substance (as DOC) was 3:1.
- Continuous darkness: yes, except during sampling and checking of the test system.

TEST SYSTEM
- Culturing apparatus: 2 litre cylindrical glass vessels.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: aeration with humidifed air.
- Measuring equipment: Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V autosampler (Shimadzu, Kyoto, Japan).
- Test performed in open system.

SAMPLING
- Sampling frequency: At the start of the test (0 h) and 3h after addition of the test substance in order to estimate any adsorption of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline by the activated sludge; and on day 1, 3, 7, 14, 21, 27 and 28.
- Sampling method: Prior to sampling the volume of the vessels was checked for evaporation and if necessary Milli-Q water was added. The medium was mixed thoroughly and a sample of 100 ml was taken. This sample was passed through a rough paper filter (S&S 604), and thereafter through a 0.45 µm filter (S&S FP 030/0.45). At least, the first 5 ml was discarded. 40 ml was used for DOC analysis. Another 40 ml was stored in a deep-freeze for possible analysis until delivery of the final report with a maximum of three months.
- Sample storage before analysis: Not applicable.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 1
- Reference substance (aniline): 1

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

Preliminary information: In Project 496991, 203278/A, the flask method applied for the determination of the water solubility of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. The water solubility of the test substance at 20°C was 1.78 g/l. The pH of the aqueous samples was 7.5. The preparation method for the Zahn-Wellens test was comparable to the preparation method in the water solubility test.

Test performance:

Preparation of test vessels:
1) Test substance mixture (1 vessel):
500 ml mineral medium
500 ml activated sludge (final concentration in test mixture: 0.75 g SS/l)
330 ml of the 10 g/l m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline filtered stock solution.
Made up to 2 litres with mineral medium. DOC of the m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline test solution was 224.3 mg/l (t=0h).
The ratio of inoculum and m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline (as DOC) was 3:1.

2) Blank control mixture (1 vessel):
500 ml mineral medium
500 ml activated sludge (final concentration in test mixture: 0.75 g SS/l)
Made up to 2 litres with mineral medium.

3) Reference substance (1 vessel):
500 ml mineral medium
500 ml activated sludge (final concentration in test mixture: 0.75 g SS/l)
702.6 mg aniline.
Made up to 2 litres with mineral medium.
DOC of this solution (315.2 mg/l) was 253.3 mg/l (t=0h).
The ratio of inoculum and aniline (as DOC) was 3:1.

Prior to sampling the volume of the vessels was checked for evaporation and if necessary Milli-Q water was added. The medium was mixed thoroughly and a sample of 100 ml was taken. This sample was passed through a rough paper filter (S&S 604), and thereafter through a 0.45 µm filter (S&S FP 030/0.45). At least, the first 5 ml was discarded. 40 ml was used for DOC analysis. Another 40 ml was stored in a deep-freeze for possible analysis until delivery of the final report with a maximum of three months.

Frequency of sampling
- At the start of the test (0 h) and 3h after addition of the test substance in order to estimate any adsorption of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline by the activated sludge;
- Day 1, 3, 7, 14, 21, 27 and 28.

Sample pretreatment
- Not applicable, samples were analysed on the day of sampling. The reserve samples were not analysed for this test.

% Degradation

Parameter:

% degradation (DOC removal)

Value:

8

Sampling time:

28 d

Details on results:

The results obtained during the test period showed no significant DOC removal in the first 3 hours of incubation in either of the test vessels indicating no or only limited adsorption to sludge during this period.

The biodegradation values calculated from the DOC measurements performed during the 28-day test period revealed no significant biodegradation of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. Thus, the biodegradation values did not reveal more than 20% biodegradation of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline during the test period.

BOD5 / COD results

Results with reference substance:

The removal of aniline was at least 70% within 14 days (98%).

Any other information on results incl. tables

2nd Acceptability criterion:

 The criterion for gradual removal of test substance during the test was not applicable as a maximum of 8% removal was observed in the test solution.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was not inherently biodegradable under the conditions of the 'Zahn-Wellens / EMPA Test' presently performed.

Executive summary:

The inherent biodegradabilty of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline under the conditions of the Zahn-Wellens/EMPA test was investigated according to OECD guideline 302 B and GLP principles. A single test concentration of 224,3 mg/L (as DOC) was tested during 28 days. Based on the obtained results, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was not inherently biodegradable. The study is considered to be reliable without restrictions.