Reaction mass of 3-(2-{3-cyanato-5-[2-(3-cyanatoaryl)heteropolycycle-5-yl]aryl}heteropolycycle-5-yl)aryl cyanate and heteropolycycle -2,5-diyldi-3,1-arylene dicyanate

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:

09 May 2011 - 26 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Sewage treatment works (Suffolk, UK), which treats predominantly domestic waste. At the time of collection, the sludge was sieved (1 mm2) then transported to the laboratory and left to stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.

- Laboratory culture:
The concentration of suspended solids in a homogenised sample was determined before the start of the test. Aliquots (10 mL) of the sludge were filtered through dried and preweighed Whatman GF/C filters, which were then dried again at approximately 105°C for one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the sludge was then determined and the volume required to give a solids level of 30 mg/L in test cultures was calculated. This was added to bottles three days before test initiation to allow a period of ageing.

- Preparation of inoculum for exposure:
Three days before test initiation, a volume (8 litres) of mineral salts medium (MSM) was prepared and the pH determined and adjusted to 7.6 with 5M HCl. Appropriate volumes of the MSM were then added to eight clear glass culture bottles (500 mL) and each was inoculated with activated sludge. Magnetic stirrer bars were added to the cultures and each bottle was fitted with an electrolytic cell assembly (containing the electrolyte, 1M copper sulfate solution, and the CO2 absorber, 5 mL of 2M potassium hydroxide) and connected to the respirometer. The magnetic stirrers were set to give a vortex in each test mixture and the instrument was initiated.

Duration of test (contact time):

28 d

Initial conc.:

50 other: mgO2/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST:
- Test substance: 2 vessels containing LZ1554 in inoculated mineral salts medium (25 mgO2/500 mL)
- Allytlthiourea test: 1 vessel containing LZ1554 in inoculated mineral salts medium (25 mgO2/500 mL) +allylthiourea (11.6 mg/L)
- Test for inhibition: 1 vessel containing LZ1554 (25 mgO2/500 mL) + sodium benzoate (25 mgO2/500 mL) in inoculated MSM

CONTROL AND BLANK SYSTEM
- Blank control: 2 vessels containing inoculated mineral salts medium
- Allylthiourea control: 1 vessel containing inoculated mineral salts medium + allylthiourea (11.6 mg/L)

REFERENCE:
- 1 vessel containing inoculated mineral salts medium + sodium benzoate (25 mgO2/500mL)

A solubility trial showed that the test substance was sufficiently soluble in acetone to prepare a stock solution. Therefore, at test initiation, the test substance was heated to ca. 80°C under a stream of nitrogen and a stock solution was prepared by weighing 213 mg and dissolving this in acetone (100 mL). An aliquot (5 mL) of the stock was added to each empty (amber glass) test bottle. The acetone was evaporated with nitrogen gas and the bottles allowed to stand for at least 1 hour. At the end of the standing period, the bottles were flushed again with nitrogen to remove any traces of solvent. The contents of the vessels prepared on Day - 3 were then transferred to the treated bottles. The final nominal test concentration was 25
mgO2/500mL or 50 mgO2/L.

To ensure similarity in preparation of all mixtures acetone (5 mL) was added to empty control and reference bottles and these were then treated as above.

Sodium benzoate (20 mL) was added as an aqueous stock solution (0.750 g/L) in MSM to the reference and inhibition assay cultures to give a final nominal concentration of 25 mgO2/500 mL or 50 mgO2/L.
A stock solution of allylthiourea (ATU) was prepared in MSM at a nominal concentration of 1.16 g/L. An aliquot (5 mL) of this stock solution was added to one culture containing inoculated MSM alone and to one containing the test substance.

The pH of the cultures was measured and adjusted to 7.4 + 0.2 with 5M NaOH.

The CO2 absorber was replaced in each cell (to ensure the maximum adsorption capacity in the test) and the cultures were sealed and returned to the water bath to equilibrate. The cells were connected to the computer-controlled system and the test was initiated.





















STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: The oxygen consumption in mixtures containing LZ1554 was negligible during the test. No consumption was measured above that in the controls

Results with reference substance:

The results obtained for the rate of degradation of sodium benzoate (60% of its Theoretical Oxygen Demand after 3 days) and for the cumulative amount of oxygen consumed by the control mixtures (30.98 and 27.18 mgO2/L) fulfilled the validity criteria for this test.

In the presence of LZ1554 the degradation of sodium benzoate achieved 61% after 4 days indicating that the test substance was not inhibitory to the microbial inoculum.

The pH of test mixtures ranged between 7.22 and 7.39 at the start of the test and 7.36 and 7.50 at the end. The temperature of the water bath ranged from 21.7 to 24.1°C.

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.10 mgO2/500 mL or 60% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 23.90 mgO2/500 mL or 96% by Day 28. In the presence of LZ1554, degradation of sodium benzoate had achieved 61% by Day 4. Cumulative levels of oxygen consumption by the controls after 28 days (15.49 and 13.59 mgO2/500 mL, equivalent to 30.98 and 27.18 mgO2/L) were considered to be acceptable for this assay system. These results confirm that LZ1554 was not inhibitory to the activity of the microbial inoculum and that the test was valid.

Oxygen consumption by the mixture containing Allylthiourea (ATU) was equivalent to 22.18 mgO2/L on Day 28 of the test. Biodegradation of the test substance in the ATU treated test mixture was 4% of the ThOD (25 mgO2/500 mL) by the end of the test (Day 28). This was not considered to be biologically significant.

Validity criteria fulfilled:

yes

Remarks:

See "any other information on material and methods" for validity criteria

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

LZ1554 is not considered to be readily biodegradable under the conditions of this test.

Executive summary:

The ready biodegradability was determined in a GLP-compliant study in accordance with OECD guideline no. 301F. LZ1554 is not considered to be readily biodegradable under the conditions of this test.

Description of key information

The information available on biodegradation revealed that the substance is not ready biodegradable. In a study according to OECD 301 F degradation reached < 1 % after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater