Reaction mass of (3R)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one and (3S)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one

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:

From February 12,2014 to March31,2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

NACET00502

IUPAC Name:

NACET00502

Constituent 2

Reference substance name:

3-Methyl-5-(2,2,3-trimethylcyclopentyl)pentan-2-one

Cas Number:

119464-63-0

Molecular formula:

C14H26O

IUPAC Name:

3-Methyl-5-(2,2,3-trimethylcyclopentyl)pentan-2-one

Test material form:

other: light yellow oil

Study design

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): On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes, and inland sea; return sludge from sewage plants).
- Laboratory culture: Activated sludge, which was prepared and controlled in this laboratory according to the test method described in OECD, was us ed in this study (sampling period: November, 2013, initiation date of use: December 20, 2013).
- Method of cultivation: The activated sludge, which was cultivated for 22 hours after the synthetic sewage was added.
- Preparation of inoculum for exposure: The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium
dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0 ± 1
- Pretreatment: Decision of additive amount of activated sludge
Additive amount of activated sludge into the test vessel was 2.51 mL on the basis of the concentration of suspended solid in the activated sludge
which was determined by the following methods:
Method In accordance with Japanese Industrial Standards (JIS) K 0102-2008 Section 14.1
Result 3590 mg/l
- Concentration of sludge: 30 mg/l

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The basal culture medium (4 L) was prepared at the same proportion as the following method; purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical) was added to each 3 ml aliquot of solutions A, B, C and D, which are described in JIS K 0102-2008 Section 21, in order to prepare 1 l of solution.
- Test temperature: 25±1°C
- pH: The pH of this solution was then adjusted to 7.0.
- pH adjusted: yes
- Continuous darkness: yes


TEST SYSTEM
- Number of culture flasks/concentration: 3

CONTROL AND BLANK SYSTEM
- Inoculum blank: In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (2.36 ml) of activated sludge from 300 ml].

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

% Degradation 1

Parameter:

other: BOD

Value:

ca. 6

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (test mat. analysis)

Remarks:

GC

Value:

ca. 37

Sampling time:

28 d

Any other information on results incl. tables

In the vessel Nos. 3 and 4, the percentage biodegradation by BOD was -3 % and -4 %, suggesting that the test item was not biodegraded. The percentage residue of the test item was good as 98 % and 97 %. From the result of the qualitative analysis for converted products by LC-MS, the converted product was not detected. Therefore, all the test item remained. Detected DOC (percentage detection: 4%) was thought to be derived from the test item dissolved in the test solution.

In vessel No. 2, the percentage biodegradation by BOD was 26 %, suggesting that some of the test item were biodegraded. The percentage residue of the test item was 0% and the peak derived from converted product was detected on the GC chromatogram. From the results of the qualitative analysis for converted products, the converted product A (2,2,3-trimethylcyclopentyl acetate) was detected. Detected DOC (percentage detection: 70 %) was thought to be derived from the converted product A dissolved in the test solution because the converted product A was detected in the sample for LC-MS -2 (for water-soluble converted products). The DOC value calculated from the theoretical amount of the converted product A was compared with experimental DOC value. As a result, the calculated DOC of 17.2 mgC was agreed with experimental value (1 6.8 mgC). Therefore, all of the test item was thought to be converted to the converted product A in the vessel No. 2, and then the produced converted product A was dissolved

in the test solution.

The percentage biodegradation by BOD was thought to be derived from the biodegradation of a part of the alkyl chain in the structural formula of the test item.

		Sludge +test item
		Vessel No. 2	Vessel No. 3	Vessel No. 4	average
Percentage biodegradation by BOD	%	26	-3	4	6
Percentage biodegradation of test item (by GC)	%	100	4	5	37

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the test conditions of this study, the test item was not biodegraded and remained in two of three test vessels. In another test vessel, the test item was completely converted and 2,2,3-trimethylcyclopentyl acetate was produced. 2,2,3-trimethylcyclopentyl acetate was not furher biodegraded.

Executive summary:

The substance has been tested for ready biodegradation according to the Regulation OECD 301C.

Conditions of incubation:

Concentration of test item: 100 mg/L

Concentration of activated sludge: 30 mg/L (as concentration of suspended solid)

Volume of test solution: 300 mL

Incubation temperature: 25±1°C

Incubation duration: 28 days (under dark conditions)

Measurement and analysis for calculation of percentage biodegradation:

a) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

b) Determination of test item by gas chromatography (GC)

Under the test conditions of this study the test item was not biodegraded and remained in two of three test vessels. In another vessel the test item was completely converted and 2,2,3-trimethylcyclopentyl acetate was produced. 2,2,3-trimethylcyclopentyl acetate was not biodegraded.