6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

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:

20 June 2012 to 19 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with OECD, EU and ISO test guidance in copliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: ISO 9439, 1999 and ISO 10634, 1995

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Hygroscopic: No
Volatile: No
Stability in water: Unknown
Solubility in water: Not indicated

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas','s-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.4 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (43 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.
Reason for selection :The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic conditions.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test concentration and preparation of test solutions: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was tested in duplicate at 15 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula.
Since 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 29.0 mg; test substance bottle B: 29.1 mg and toxicity control bottle: 29.1 mg). To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

Reference Substance
Identification number: RS186
Name: Sodium acetate
Description: White powder (determined at NOTOX)
Molecular formula: CH3COONa (taken from label)
Molecular weight: 82.03 (taken from label)
Batch number: K34333668
Article number: 1.06268.0250
Purity: ≥99.0%
Expiry Date: 28 February 2015
Certified: Yes
Storage conditions: At room temperature in the dark
Supplier: Merck, Darmstadt, Germany

Reference substance concentration and preparation of test solutions: A solution of sodium acetate was prepared by dissolving 400.1 mg in Milli-RO water and making this up to a total volume of 100 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/l).

Test procedure and conditions
Test duration: 28 days (last CO2-measurement on the 29th day). During the test period the test media were aerated and stirred continuously.
Test vessels: 2 litre all-glass brown coloured bottles.
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.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) 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.
Type and number of 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).
Preparation: At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial organisms and mineral components.
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 and recording:
pH: At the start of the test (day 0) and on the 28th day.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

2 - 12

Sampling time:

28 d

Details on results:

The ThCO2 of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was calculated to be 2.99 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed 2 and 12% biodegradation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, 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% biodegradation occurred within 14 days (33%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Results with reference substance:

The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

Notes: Except for the percentages biodegradation, all calculations are performed without rounding off. Produced CO₂: negative values are expressed as 0.00 ml HCI.

 

CO₂production and percentage biodegradation of the positive control substance

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Positive control
2	48.33	39.90	8.43	9.3	9.3	11
5	47.28	24.42	22.86	25.1	34.4	40
7	46.51	35.63	10.88	12.0	46.4	54
9	47.44	40.62	6.82	7.5	53.9	63
14	45.71	39.24	6.47	7.1	61.0	71

¹⁾Calculated as the ratio between CO₂produced (cumulative and the ThCO₂of sodium acetate 85.6 mg CO₂/2l

 

CO₂production and percentage biodegradation of the test substance (bottle A)

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Bottle A
2	48.33	48.83	0.00	0.0	0.0	0
5	47.28	47.26	0.02	0.0	0.0	0
7	46.51	46.51	0.00	0.0	0.0	0
9	47.44	47.06	0.38	0.4	0.4	1
14	45.71	45.67	0.03	0.0	0.5	1
19	44.28	44.68	0.00	0.0	0.5	1
23	43.71	44.29	0.00	0.0	0.5	1
27	43.95	44.75	0.00	0.0	0.5	1
29	46.11	45.51	0.60	0.7	1.1	1
29	48.81	48.17	0.63	0.7	1.8	2
29	50.18	49.90	0.28	0.3	2.1	2

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 86.7 mg CO₂/2l

 

CO₂production and percentage biodegradation of the test substance (bottle B)

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Bottle B
2	48.33	48.79	0.00	0.0	0.0	0
5	47.28	46.98	0.30	0.3	0.3	0
7	46.51	45.95	0.56	0.6	0.9	1
9	47.44	45.91	1.53	1.7	2.6	3
14	45.71	45.10	0.60	0.7	3.3	4
19	44.28	43.15	1.13	1.2	4.5	5
23	43.71	42.45	1.26	1.4	5.9	7
27	43.95	42.98	0.97	1.1	7.0	8
29	46.11	43.98	2.13	2.3	9.3	11
29	48.81	47.58	1.23	1.3	10.7	12
29	50.18	50.17	0.01	0.0	10.7	12

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 87.0 mg CO₂/2l

 

CO₂production and percentage biodegradation of toxicity control

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1)(%)
	Blank (mean)	Toxicity control
2	48.33	41.84	6.49	7.1	7.1	4
5	47.28	21.68	25.60	28.2	35.3	20
7	46.51	35.05	11.46	12.6	47.9	28
9	47.44	39.53	7.91	8.7	56.6	33
14	45.71	50.00	0.00	0.0	56.6	33

¹⁾Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test substance and positive control: 172.6 mg CO₂/2l (ThCO₂test substance: 87.0 mg CO₂/2l + ThCO₂sodium acetate: 85.6 mg CO₂/2l)

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol.

 

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard 9439, 1999 and ISO Standard 10634, 1995. The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was tested in duplicate at 15 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was calculated to be 2.99 mg CO2/mg.

 

The study consisted of six bottles:

-2 inoculum blanks (no test substance),

-2 test bottles (6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol),

-1 positive control (sodium acetate) and

-1 toxicity control (6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol plus sodium acetate).

 

Since 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface. Test duration was 28 days (last CO2-measurement on the 29thday).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 2 and 12% biodegradation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, 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, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was designated as not readily biodegradable.

Description of key information

Key value determined by experimental conditions utilising OECD, EU & ISO test guidance.

The substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The relative biodegradation values calculated from the measurements performed during the test period revealed 2 and 12% biodegradation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not readily biodegradable under the conditions of the modified Sturm test presently performed.