[No public or meaningful name is available]

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:

2 August 2012 - 26 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms” stipulated in the “Testing Methods for New Chemical Substances”

Version / remarks:

31 March 2011, including the latest revisions

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanol, 2-methyl-, reaction products with 1,5-diisocyanatopentane

Cas Number:

1357171-37-9

Molecular formula:

not applicable (a generic formula cannot be provided for this UVCB substance)

IUPAC Name:

1-Propanol, 2-methyl-, reaction products with 1,5-diisocyanatopentane

Test material form:

liquid

Details on test material:

- Physical appearance: colorless and transparent liquid
- Storage conditions: the test sample was sealed with nitrogen and stored by cold and dark place with silica gel.

Specific details on test material used for the study:

Stability: Reacts with water; reacts when heated; stability in light is not investigated.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: sludge consisted of surface water containing the surface soil of rivers, lakes and inland sea, and returned sludge from a sewage-treatment plant. The sludge was collected from 10 different locations in Japan. Date of collection: May, 2012
- Preparation of sludge: activated sludge was prepared and managed at our facilities and used in the test. In the test, synthetic sewage (glucose, peptone and potassium dihydrogen phosphate were dissolved in purified water, and pH was adjusted to 7.0 ±1.0) was added to the solution, and thus activated sludge was obtained. After 18.5 hours, the activated sludge was used. Date of use: 18 June 2012.
- Concentration of sludge: 3250 mg/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

100 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

43.5 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: In accordance with Japanese Industrial Standerds (JIS)K 0102-2008 Section 14.1
- Test temperature: 25 ±1°C
- pH (at the end of the test): 6.7 in abiotic control and 7.3 in test solution
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: glass erlenmeyer flask
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: not indicated
- Measuring equipment: closed-system oxygen consumption measuring apparatus:
* Temperature controlled batch: AI-0001 (Asahi Techneion Co.)
* Data sampler: OM7000A (Ohkura Electric Co.)
* Vessel: glass bottles
- Details of trap for CO2: soda lime No. 1 (Wako Pure Chemical Industries) was used to absorb CO2
- Other: test solutions were stirred continuously by a stirrer

SAMPLING
- Sampling frequency: BOD of the test solutions was continuously measured
- Sampling method: by use of closed-system oxygen consumption measuring apparatus
- Other: The incubation temperature and the conditions of the test solutions were measured and recorded once a day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, one replicate
- Abiotic sterile control: yes, one replicate (water + test item)
- Toxicity control: no
- Other: one positive control containing sludge and the reference substance

STATISTICAL METHODS: no statistical analysis was performed

Reference substance

Reference substance:

aniline

Remarks:

ThOD: 72.3 mg/L

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: mean of 3 replicates was -1%

Details on results:

- Biodegradation was calculated by BOD. The BOD in individual test flasks was 6.9, 6.8 and 7.6 mg after 28 days. Biodegradation in individual flasks was -2, -2 and 0%

- Visual inspections showed that the test solutions were colorless. An insoluble compound (not sludge) was observed. Growth of the sludge was not observed.

- HPLC analysis of the test item showed that 100% of the test item was decreased after 28 days. A qualitative analysis was carried out to identify insoluble converted products.
- It was presumed that the test item in both the abiotic control and the test solution (sludge + test item) went through the hydrolysis of the isocyanate group (N=C=O) to form urea bonds [-NH-C(=O)-NH-] among molecules and turned into three-dimensional polymer having a molecular weight of not less than 1000 that was insoluble in general solvents. It is presumed that the formed three-dimensional polymer remained without undergoing biodegradation.

BOD5 / COD results

Results with reference substance:

The reference substance was biodegraded for 94% after 14 days.

Any other information on results incl. tables

Table 1 Biodegradation calculated by BOD

Vessel	Day 7		Day 14		Day 21		Day 28		Mean
	BOD (mg)	Deg. (%)	BOD (mg)	Deg (%)	BOD (mg)	Deg (%)	BOD (mg)	Deg. (%)	DEG. (%)
Sludge + aniline	54.7	70	73.9	94	76.4	95	77.9	97
Control blank	4.4		6.1		7.6		7.8
Sludge + test item	4.8	1	6.2	0	6.8	-2	6.9	-2	-1
Sludge + test item	4.5	0	6.0	0	6.8	-2	6.8	-2
Sludge + test item	4.4	0	6.5	1	7.4	0	7.6	0
Water + test item	0.0		0.0		0.5		0.6

Deg. = [BOD-B]/[TOD]*100%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks' section

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

D-370N did not biodegrade over a period of 28 days in the MITI test (mean biodegradation of -1%).

Executive summary:

In a readily biodegradation study, performed as a MITI test according to JIS K 0102 -2008, D-370N was evaluated for its ability to biodegrade over a period of 28 days. The test item was tested in triplicate alongside an abiotic control, a blank control and a reference control (aniline). Biodegradation was determined by continuous measurement of the biological oxygen demand (BOD) of the test item using a closed-system oxygen consumption measuring apparatus. Soda lime was used to absorb carbon dioxide. After the end of the incubation, the test item in the test solutions was analyzed, and the converted products that were difficult to determine quantitatively were determined qualitatively by HPLC and LC-MS. Furthermore, although it was presumed that the test item would not be dissolved in the testing solutions, analysis was performed as to the dissolved organic carbon (DOC) in the test solutions to confirm whether or not there was a formation of water-soluble converted products. Results showed that the test item was biodegraded for -1% (mean of three replicates). The TOD was calculated to be 43.5 mg while the mean BOD was determined to be 6.8 mg. Analyis showed a mean DOC of 0.87. Since the test item did not dissolve in water at a test concentration (100 mg/L) or higher, the DOC percentage biodegradation was not calculated. HPLC analysis of the test item showed that 100% of the test item was decreased after 28 days. The insoluble converted products were determined to be three-dimensional polymers generated by urea bonds. It was presumed that the three-dimensional polymer remained without undergoing biodegradation. The reference item was biodegraded for 94% after 14 days, showing that the test system functioned properly. The acceptability criteria were met and the test was considered to be valid.