2,6,6-trimethylcyclohexa-1,3-dienecarbonitrile

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:

May 29, 2015 to September 22, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Note: Optional toxicity control was not performed

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Note: Optional toxicity control was not performed

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Remarks:

Note: Optional toxicity control was not performed

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance for the implementation of REACH, Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance, November 2014 (version 2.0)

Deviations:

no

Remarks:

Note: Optional toxicity control was not performed

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,6,6-trimethylcyclohexa-1,3-dienecarbonitrile

EC Number:

917-351-9

Cas Number:

72152-84-2

Molecular formula:

C10H13N

IUPAC Name:

2,6,6-trimethylcyclohexa-1,3-dienecarbonitrile

Constituent 2

Reference substance name:

Safranitrile

IUPAC Name:

Safranitrile

Details on test material:

- Name of test material (as cited in study report): Safranitrile
- EC Name: 2,6,6-trimethyl-1,3-cyclohexadiene-1-carbonitrile
- CAS Number: 72152-84-2
- Physical state: yellow liquid
- Lot/batch No.: SC00012001
- Expiration date of the lot/batch: July 23, 2018

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of activated sludge: Domestic sewage works, Bois-de-Bay, Satigny, Switzerland.
- Sludge was collected in the morning, washed three times in the mineral medium by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium.
- The sludge was kept under aerobic conditions and used the same day.

Duration of test (contact time):

60 d

Initial test substance concentration

Initial conc.:

30 mg/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

APPARATUS
- Respirometer: Oxitop Control System

WATER
- Deionised water containing less than 10 mg/L dissolved organic carbon was used during the study.

STOCK SOLUTIONS
- Solution A: KH2PO4 8.5 g; K2HPO4 21.75 g, Na2HPO4.2H2O 33.4 g; NH4Cl 0.5 g (dissolved in water and made up to 1 L).
- Solution B: CaCl2 27.5 g (dissolved in water and made up to 1 L).
- Solution C: MgSO4.7H2O 22.5 g (dissolved in water and made up to 1 L).
- Solution D: FeCl3.6H2O 0.25 g; concentrated HCl one drop (dissolved in water and made up to 1 L).

MINERAL MEDIUM
- Solution A (50 mL) and deionised water (2000 mL) were mixed and Solution B (5 mL), Solution C (5 mL) and Solution D (5 mL) added before making up to 5 L with deionised water.

DETERMINATION OF DRY WEIGHT OF SUSPENDED SOLIDS
- Two 50 mL samples of the homogenised sludge were taken and water was evaporated on a steam bath, dried in an oven at 105-110 °C for two hours, and weighing the residue.

FLASK PREPARATION
- Test substance samples (13.10 mg, corresponding to 30 mg/L in 437 mL) were weighed in small aluminium boats and added to the contents of the test flasks of the Oxitop.
- For reference substance samples 43.70 mg (corresponding to 100 mg/L in 437 mL of test medium) were weighed in small aluminium boats and added to the contents of the test flasks of the Oxitop.
- Flasks were filled with 432 mL of mineral medium.
- Suspended sludge diluted to a concentration of 2.62 g/L dry matter was added.
- pH of each flask was not measured but assumed to be the same as the mineral medium.
- Two sodium hydroxide pellets were placed in the quivers on top of the bottle.
- The flasks were then closed tightly with the measuring heads and allowed to equilibrate to the test temperature.
- After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

TEST CONDITIONS
- Temperature (21.3°C - 22 °C) and stirring were checked daily.
- Oxygen consumption of each flask was recorded daily.

NOMINAL CONCENTRATIONS
- Test material: 30 mg/L
- Reference substance: 100 mg/L

TOXICITY CONTROL
- An optional toxicity control was not performed. The validity of the study was not adversely affected.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

- The activity of the inoculum was verified.
- The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled.

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

60 d

Details on results:

Safranitrile undergoes no biodegradation after 28 days in the test conditions.

BOD5 / COD results

Results with reference substance:

Biodegradation of reference substance sodium benzoate was 73 % (day 7) and 82 % (day 14). For details see Appendix 4 of attached study report 15 - E070.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Safranitrile undergoes no biodegradation after 28 days in the test conditions. Thus, Safranitrile should be regarded as not readily biodegradable according to this test.

Executive summary:

The Ready Biodegradability of Safranitrile was determined by the Manometric Respirometry Test.

Safranitrile undergoes no biodegradation after 28 days in the test conditions. Safranitrile did not significantly inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Thus, Safranitrile should be regarded as not readily biodegradable according to this test.