2-Propenoic acid, 1,1'-[7,13-dimethyl-10-(3-methyl-11-oxo-2,4,7,10-tetraoxatridec-12-en-1-yl)-10-[[2-[(9-oxo-9H-thioxanthen-2-yl)oxy]acetyl]amino]-3,6,8,12,14,17-hexaoxanonadecane-1,19-diyl] ester

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From December 19, 2002 to March 11, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Method for Testing Bio-degradability of Chemical Substances by Microorganisms stipulated in the "Testing Method for New Chemical Substances" (1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatsu No.6l5, Pharmaceutical Affai

Deviations:

no

GLP compliance:

yes

Remarks:

This study was conducted in compliance with the GLP Chapter XI stipulated in the Order Prescribing the Items of the Test Relating to New Chemical Substances Article 4,

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

IUPAC Name:

2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Constituent 2

Reference substance name:

86273-46-3

Cas Number:

86273-46-3

IUPAC Name:

86273-46-3

Constituent 3

Reference substance name:

2-(2'-Vinyloxyethoxy)ethyl acrylate

IUPAC Name:

2-(2'-Vinyloxyethoxy)ethyl acrylate

Constituent 4

Reference substance name:

-

EC Number:

451-690-9

EC Name:

-

Details on test material:

- Name of test material (as cited in study report): 2-(2'-Vinyloxyethoxy)ethyl acrylate
- Molecular weight (if other than submission substance): 186.21
- Substance type: clear and colorless liquid
- Physical state: liquid
- Analytical purity: 99.9% GC Method
- Lot/batch No.: FX-2TOI-B-VA
- Stability under test conditions: Stability of the test substance was tested by measurement with an infrared spectrophotometer before and after the test.
- Storage condition of test material: Store at cool temperature and light shielding.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:

PHYSICO-CHEMICAL PROPERTIES
- Solubility in water: 13 g/L(30°C)
- Solubility in Methanol: soluble
- Solubility in Acetonitrile: soluble
- Stability of test material at room temperature: Stability of the test substance was tested by measurement with an infrared spectrophotometer before and after the test. An infrared absorption spectrum of the test substance obtained after the test was compared with that obtained before the test, and there was no significant difference between the two infrared absorption spectra. Both spectra showed specific absorption peaks at 2880,1730, 1620 and 1200 cm-l . As described previously, the test substance was stable under the storage conditions specified, during the study period.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A mixed population of sewage sludge micro-organisms was obtained on 7 October 2008 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture:
Standard activated sludge used on the study was purchased from Chemicals Evaluation and Research Institute, Japan on January 23, 2003.

- Method of cultivation:
Activated sludge was cultured as follows. About 1 L of activated sludge was aerated in the cultivation tank for 23.5 hours. About 30 minutes after ceasing aeration, supernatant corresponding to about 1/3 of the whole volume (approximately 330 mL) was removed. Then an equal volume of 0.1% synthetic sewage was added to the remaining portion and aerated again. This procedure was repeated once every day.

- conditions for cultivation:
Stirring at 25±1 °C carried out cultivation for 28 days with a Coulometer and the quantity of oxygen consumption was measured continuously. After cultivation, residual amounts of the test substance were analyzed.

- Storage length:
28 days

- Preparation of inoculum for exposure:
not stated

- Pretreatment:
not stated

- Concentration of sludge:
A test concentration of 30 mg carbon/l was employed in the study.

- Initial cell/biomass concentration:
Not stated

- Water filtered:
yes

- Type and size of filter used, if any:
ilter paper (2S:Advantec)

Duration of test (contact time):

0 - 28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

test mat. analysis

Parameter followed for biodegradation estimation 3

Parameter followed for biodegradation estimation:

TOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium:

Culture Medium


Preparation of culture medium: Each 6 mL of solution A, B, C and D, which are prescribed in the Japanese Industrial Standard (JIS) KOI02-21, were made up to 2.0 L with pure water.

- Additional substrate:
Not applicable

- Solubilising agent (type and concentration if used):
Not applicable

- Test temperature:
The temperature of the cultivation room during the test was kept at 24.6-25.2°C, which met the study criteria (25.0 ± 1.0°C).

- pH:
Before and after the test, the pH of test solutions were measured and determined to be 7.7. The pH of the solutions in test bottles is shown in Table 1 (see attachment 1).

- pH adjusted:
not stated

- Dissolved Oxygen concentration::
7.4 mg/L

- Aeration of dilution water:
Not stated

- Sedimentation ratio of the activated sludge:
18 %

- Concentration of suspended solids:
5026mg/L, Concentration of suspended solids is within ± 1000 mg/L difference from the initial concentration.

- Continuous darkness:
not stated

- Other:


TEST SYSTEM

Treatment procedure of test solution:
To the contents of the test bottles No.1, 2, 3, 4, and 6, 90 g of sodium chloride and 90 mL of ethyl acetate were added and mixed for 10 minutes using a magnetic stirrer (speed 6). The mixtures were then permitted to stand for 10 minutes at room temperature. The ethyl acetate layer was separated by filter paper (2S:Advantec), and was diluted in ethyl acetate to make exactly 100 mL. One milliliter of this solution was sampled, and then 2 mL of ethyl acetate was added. A 2 µL aliquot of the resulting solution was injected into the GC system.

Preparation of VEEA standard solutions:
A 10.0 mg aliquot of the test substance was accurately weighed and dissolved in acetonitrile to make exactly 100 mL, and used to provide a 100 mg/L standard solution. This solution was diluted with acetonitrile to provide concentrations of 50, 20, 10 and 5 mglL, and these were used as the test substance standard solutions.

Preparation of test solutions:
The following solutions were prepared in test bottles and adjusted at test temperature.
(I) 100 mg/L test substance in pure water: bottle No.1
(2) 100 mg/L test substance in basal culture medium: bottle No. 2, 3, 4
(3) 100 mg/L aniline in basal culture medilll11: bottle No. 5
(4) Basal culture medilll11 for blank test:bottle No. 6

Preparation of test system:
A 30.0 mg aliquot of the test substance was added to 300 mL of pure water and basal culture medium (activated sludge added) to prepare the recovery samples. (These samples were treated as described in the" treatment procedures of test solution" (see above). The concentrations of the recovery samples were calculated from the standard solution, and used to calculate recovery rates.

The concentrations of the test substance in test bottles were revised from the
following equation:
Concentration of the test substance in test bottles (mg/L) = Concentration of GC sample (mg/L) x 1001 recovery rate x 300/280

Each sample was assayed once. The concentrations of the test substance were calculated.


Calibration curves were developed on the standard solution of VEEA ranging in concentration from 5-100 mg/L,
linearity (r) and intra-assay precision (CV) of the calibration curve were confirmed as:
r =0.999986,
CV = 0.4-2.5% [Table 2 and Fig. 3 in attachments 1 and 2].

The means of recovery rate (n=3) were 92.0% (CV=0.7%) for pure water and 91.9%(CV=1.2%) for basal culture medium (sludge was added), respectively% [Table 3].

Therefore, it was thought that this analysis method was suitable to measure the concentration of VEEA.

In the pure water (bottle No.l) at the end of the experiment period, two degradation products were detected. Although the mass spectra were measured, the data to identify was not obtained.

SAMPLING
- Sampling frequency:
Not stated

- Sampling method:
Not stated

- Sterility check if applicable:
Not stated

- Sample storage before analysis:
Not stated


CONTROL AND BLANK SYSTEM
- Inoculum blank:
Not stated

- Abiotic sterile control:
Not stated

- Toxicity control:


STATISTICAL METHODS:

The calibration curve was obtained by the least squares method, using the relationship between peak area (y) of the test substance and nominal concentration (x).

Equation for calibration curve: y = ax + b
Where:
x is nominal concentration,
y is peak area of the test substance.
Concentration of the test substance was calculated from the following equation;
Concentration (mg/L) = y/a - b/a

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Parameter:

% degradation (TOC removal)

Value:

> 84.4

Sampling time:

28 d

Remarks on result:

other: Mean of 3 values 84.7, 85.8 and 82.6

BOD5 / COD results

Results with reference substance:

It was calculated from the BOD that the percentage biodegradation of reference substance on the 7th day was 61.5%, on the 14th day was 71.5%. Therefore, it was confirmed that this test is valid.

Any other information on results incl. tables

Discussion and Conclusion:

The mean percentage biodegradation of VEEA calculated by BOD, TOD and GC analysis was 82.1, 84.4 and 100.0%, respectively.

In the pure water (bottle number 1) at the end of the experiment period, test substance (VEEA) and its degradation products were detected (retention time; approximately 10.2 and 13.6 min.). And test substance and its degradation product was not detected in the sludge series (bottle No.2-4). However, it was suggested that degradation products were produced about 16% from TOC analysis.

In addition, the BOD curve at the end of the experiment period showed the loose rise, and biodegradation was continued succeedingly within the test bottles. It was thought that these degradation products also received biodegradation gradually, and disappeared.

Although the test substance is decomposed in water, it was judged that the test substance is biodegraded.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

It was calculated from BOD that the percentage biodegradation of reference substance on the 7th day was 61.5%, on the 14th day was 71.5%. Therefore, it was confirmed that this test is valid.

Interpretation of results:

readily biodegradable

Conclusions:

The mean percentage biodegradation of VEEA calculated by BOD, DOC and GC analysis was 82.1, 84.4 and 100.0%, respectively. Although the test substance is decomposed in water, it was judged that the test substance is biodegraded.

Executive summary:

Examination overview:

(l)Test concentration Test substance: l00 mg/L

Active sludge: 30 mg/L

(2) Experiment period 28 days (From January 27. 2003 to February 24, 2003)

(3) Results:

Method test bottle No.2 test bottle No.3 test bottle No.4 test bottle Mean

% Biodegradation by BOD 81.7 85.2 79.3 82.1

% Biodegradation by DOC 84.7 85.8 82.6 84.4

% Biodegradation by GC 100 100 100 100

Conclusion:

The mean percentage biodegradation of VEEA calculated by BOD, DOC and GC analysis was 82.1, 84.4 and 100.0%, respectively. Although the test substance is decomposed in water, it was judged that the test substance is biodegraded.