A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

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:

From June 25, 2008 to August 21, 2008

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution 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): freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.1 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (35 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium. Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Duration of test (contact time):

29 d

Initial conc.:

17.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Stock solutions of mineral components:
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

Mineral medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

TEST SYSTEM
Type and number of bottles: 2-liter all glass coloured brown bottles
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. 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.


Measurements
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.
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. Phenolphthalein (1% solution in ethanol, Merck, Germany) was used as pH-indicator.
On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck, Darmstadt, Germany) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

- test temperature: 21-24°C
- pH - 7.6 - 8.0

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

> 7 - < 15

Sampling time:

29 d

Details on results:

Theoretical CO2 production
The ThCO2 of the test substance was calculated to be 2.09 mg CO2/mg.
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.

The relative degradation values calculated from the measurements performed during the test period revealed 15 and 7% degradation of the test substance, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control more than 25% degradation occurred within 14 days (45%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Results with reference substance:

74% degradation after 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the test substance was not readily biodegradable.

Executive summary:

A study was conducted to determine biodegradability of the test substance according to OECD Guideline 301B, EU Method C.4-C and ISO 9439, in compliance with GLP. The test substance (95% purity) was tested in duplicate at 35 mg per 2 L, corresponding to 10 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) was calculated to be 2.09 mg CO2/mg. Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, it was weighed on glass slides and quantitatively added to the test bottles (2 L) containing medium with microbial organisms and mineral components. The source of the inoculum was an activated sludge freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage. Sodium acetate was used as the reference substance at 40 mg/L. The solutions were continuously stirred to ensure optimal contact between the test substance and the test organisms. The relative degradation values calculated from the measurements performed during the test period revealed 15 and 7% degradation of test substance, for the duplicate bottles. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, the test substance was found not to inhibit microbial activity. The reference substance attained 74% biodegradation by Day 14. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the test substance was not readily biodegradable (Desmares-Koopmans, 2008).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A study was conducted to determine biodegradability of the test substance according to OECD Guideline 301B, EU Method C.4-C and ISO 9439, in compliance with GLP. The test substance (95% purity) was tested in duplicate at 35 mg per 2 L, corresponding to 10 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) was calculated to be 2.09 mg CO2/mg. Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, it was weighed on glass slides and quantitatively added to the test bottles (2 L) containing medium with microbial organisms and mineral components. The source of the inoculum was an activated sludge freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage. Sodium acetate was used as the reference substance at 40 mg/L. The solutions were continuously stirred to ensure optimal contact between the test substance and the test organisms. The relative degradation values calculated from the measurements performed during the test period revealed 15 and 7% degradation of test substance, for the duplicate bottles. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, the test substance was found not to inhibit microbial activity. The reference substance attained 74% biodegradation by Day 14. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the test substance was not readily biodegradable (Desmares-Koopmans, 2008).