N,N''-(methylenedi-4,1-phenylene)bis[N'-(4-methylphenyl)urea]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 1993 - 23 December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Stable in water for at least 96 hours.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Dommel', 's-Hertogenbosch, The Netherlands.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Storage length: not indicated
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted was used as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: mineral components, Milli-Q water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This misxture was aerated wotj CO2-free air overnight to purge the system of CO2.
- Concentration of sludge: 4.5 g suspended solids per litre in the concentrated sludge
- Water used: tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to OECD 301 guideline
- Test temperature: 21 - 23°C.
- pH: start: 7.5; end: 7.6-7.9
- pH adjusted: no
- Aeration of dilution water: continuously, except for day 19, 23 and 25 where the aeration was temporarely decreased or ceased (this was cionsidered to have no effect on the integrity of the study).
- Suspended solids concentration: 10 mL supernatant liquid/L (see 'Details on inoculum' section for details)
- Continuous darkness: not indicated

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with CO2 free air (mixture of 21% oxygen and 79% nitrogen).
- Measuring equipment: The CO2 produced in each test bottle reacted with barium hydroxide in a gas scrubbing bottle and was precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl. Phenolphthalein was used as pH-indicator.
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for all bottles tested. On the 28th day, samples were removed from each bottle to enable the possibility for DOC analysis.
- Sampling method: titration

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: no
- Toxicity control: yes, 1 replicate

STATISTICAL METHODS: none

Reference substance:

acetic acid, sodium salt

Remarks:

Batch 049 TA933768, TOC 12 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1.5

Sampling time:

28 d

Remarks on result:

other: Results is based on the mean value of % degradation seen in duplicate bottles tested (1.7% and 1.2%).

Details on results:

ThCO2 of the test item was calculated to be 2.75 mg CO2/mg. ThCO2 of the reference item (sodium acetate) was calculated to be 1.073 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of test substance (1.7% and 1.2%, based on ThCO2). Since the test substance was hardly soluble in water, the lack of microbial degradation could be a consequence of the limited availability of KY-AF for micro-organisms.
In the toxicity control, more than 25% biodegradation occurred in 14 days (28.6%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see attached illustration).
For tables containing full results, see attached background material (Tables.png).

Results with reference substance:

71% biodegradation in 14 days, 76% biodegradation in 28 days.

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria, see 'Overall remarks' section.

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of the modified Sturm test, according to OECD 301B guidelines, the test item was found to biodegrade for 1.7% and 1.2% respectively for the two replicates tested, after 28 days of incubation. Therefore, it is not considered to be readily biodegradable.

Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and GLP principles, the test item did not reach the pass level of 60% for ready biodegradability, neither within the 10 -d window nor after 28 days of incubation. Therefore, it is designated as not readily biodegradable. All the criteria for acceptability of the test were met, so the study is considered valid without restrictions.

Description of key information

The results of a reliable study, performed according to OECD guideline 301B and GLP principles, showed that the substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

In a test performed according to OECD 301B (modified Sturm test) and GLP principles, the test item did not reach the pass level of 60% for ready biodegradability, neither within the 10-d window nor after 28 days of incubation. Therefore, it is designated as not readily biodegradable. All the criteria for acceptability of the test were met, so the study is considered valid without restrictions.