Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

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:

22 September 2015 to 22 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Exact amounts of the test substance were weighed form a sub stock. This weighing was performed under light conditions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Exact amounts of the test substance were weighed form a sub stock. This weighing was performed under light conditions.

GLP compliance:

yes

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 determined to be 3.9 g/l in the concentrated sludge. Before use, the sludge was allowed to settle (34 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 items under aerobic conditions.

Duration of test (contact time):

28 d

Initial conc.:

1 g/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Reference item concentration and preparation of test solutions
A solution of sodium acetate was prepared by dissolving 401.2 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 concentration and preparation of test solutions
Phenol, 1,1-dimethylpropyl derivs. was a colourless to pale yellow solid and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l. A sample of the test substance was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma, Steinheim, Germany) as total carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as inorganic carbon (IC) standard. The Total Organic Carbon (TOC) content of the test substance was determined to be 81%. The test substance was tested in duplicate at a concentration of 15 mg/l, corresponding to 12 mg TOC/l. No correction was made for the purity/composition of the test substance. Before use the test substance was heated in a water bath (90°C) to liquefy and homogenize and sub stocks were weighed for several projects. Sub stocks were stored under nitrogen in the dark. On the day of testing weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 30.5 mg; test item bottle B: 30.3 mg and toxicity control bottle: 30.0 mg). An amount of the test substance was weighed form a sub stock. This weighing was performed under light conditions. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. The time between weighing and introduction to the test vessels was maximally 35 minutes, the time required for handling. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and test medium.
Test procedure and conditions
Test duration: 28 days (last CO2 measurement on day 29). During the test period, the test media were aerated and stirred continuously.

Test vessels: 2 litre glass brown coloured bottles. Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon. 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-RO water and made up to 1 litre, pH 7.4 ± 0.2 B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre. C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre. D) 0.25 g FeCl3.6H2O dissolved in Milli-RO 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), 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).

Illumination: The test media were excluded from light.

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of 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 item and inoculum (2 bottles).

Inoculum blank: containing only inoculum (2 bottles) Positive control: containing reference item and inoculum (1 bottle). Toxicity control: containing test item, reference item and inoculum (1 bottle).

Preparation: At the start of the test (day 0), test and reference item 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.Determination of CO2 Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and 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 (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of 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) was used as pH-indicator. On day 28, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck) 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. Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.Measurements and recording pH: At the start of the test (day 0) and on day 28, before addition of concentrated HCl.

Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 1 - <= 8

Sampling time:

28 d

Details on results:

Theoretical CO2 production
The ThCO2 of Phenol, 1,1-dimethylpropyl derivs. was calculated to be 2.96 mg CO2/mg.Biodegradation

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Phenol, 1,1-dimethylpropyl derivs. (8% and 1%, based on ThCO2). In the toxicity control, more than 25% biodegradation occurred within 14 days (35%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.Monitoring of temperatureThe temperature recorded in a vessel with water in the same room varied between 21.8 and 22.8°C.

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 item sodium acetate, which showed a normal biodegradation curve.

pH values of different test media

Test medium:	At the start of the test:	On day 28:
Blank control (A)	7.9 →^17.6	7.6
Blank control (B)	7.7 →^17.6	7.6
Positive control	7.7 →^17.6	7.6
Phenol, 1,1-dimethylpropyl dervis. (A)	7.7 →^17.6	7.6
Phenol, 1,1-dimethylpropyl dervis. (B)	7.7 →^17.6	7.6
Toxicity control	7.7 →^17.6	7.6

¹-Adjusted using 1 M HCI

 

Notes: Except for the percentages biodegradation, all calculations are performed without rounding off.

Produced CO₂: Negative value are expressed as 0.00 ml HCI.

 

HCI titrated in duplicate blank bottles

Day	HCI (0.05 N) titrated (ml)
	Blank A	Blank B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	46.75 44.71 44.68 45.33 46.23 44.48 42.31 42.53 43.66 47.05 48.97	46.96 45.78 45.34 46.49 45.32 42.56 42.31 44.23 43.91 46.36 48.84	46.86 45.25 45.01 45.91 45.78 43.52 42.31 43.38 43.79 46.71 48.91

 

HCI titrated in Ba(OH)2 solution (background bottles)

Day	HCI (0.05 N) titrated (ml)
	Bottle A	Bottle B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	49.45 49.22 47.37 50.57 50.00 51.38 49.29 48.61 49.34 48.73 48.76	48.34 49.86 48.54 48.99 50.72 49.23 47.43 49.58 50.25 48.70 49.55	48.90 49.54 47.96 49.78 50.36 50.31 48.36 49.10 49.80 48.72 49.16

 

CO2 production in the blank

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)`	Produced CO2 (mg)	Cumulative CO2 (mg)
	Ba(OH)2^1)	Blank (mean)
2 5 7 9 14 19 23 27 29 29 29	48.90 49.54 47.96 49.78 50.36 50.31 48.36 49.10 49.80 48.72 49.16	46.86 45.25 45.01 45.91 45.78 43.52 42.31 43.38 43.79 46.71 48.91	2.04 4.29 2.94 3.87 4.59 6.79 6.05 5.72 6.01 2.01 0.25	2.2 4.7 3.2 4.3 5.0 7.5 6.7 6.3 6.6 2.2 0.3	2.2 7.0 10.2 14.5 19.5 27.0 33.6 39.9 46.5 48.7 49.0

¹⁾-“Strength” of untreated 0.0125 M Ba(OH)₂solution.

 

CO₂production and percentage biodegradation of the positive control item

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (ml HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Positive control
2 5 7 9 14	46.86 45.25 45.01 45.91 45.78	30.18 19.28 36.06 37.91 41.11	16.68 25.97 8.95 8.00 4.67	18.3 28.6 9.8 8.8 5.1	18.3 46.9 56.7 65.5 70.7	21 55 66 76 82

¹⁾– Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 85.9 mg CO₂/2l

 

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

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (mg HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle A
2 5 7 9 14 19 23 27 29 29 29	46.86 45.25 45.01 45.91 45.78 43.52 42.31 43.38 43.79 46.71 48.91	45.16 43.48 46.06 46.35 46.50 44.67 43.00 43.06 44.76 46.25 46.98	1.70 1.77 0.00 0.00 0.00 0.00 0.00 0.32 0.00 0.45 1.93	1.9 1.9 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.5 2.1	1.9 3.8 3.8 3.8 3.8 3.8 3.9 4.2 4.2 4.7 6.8	2 4 4 4 4 4 4 5 5 5 8

¹⁾–Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 90.3 mg CO₂/2l

 

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

Day	HCI (0.05 N) titrated (ml)		Produced CO2 (mg HCI)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle B
2 5 7 9 14 19 23 27 29 29 29	46.86 45.25 45.01 45.91 45.78 43.52 42.31 43.38 43.79 46.71 48.91	47.06 46.85 45.80 46.61 47.51 43.65 43.98 43.48 44.67 46.69 48.23	0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.68	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8	0 0 0 0 0 0 0 0 0 0 1

¹⁾–Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 89.7 mg CO₂/2l

 

CO₂produced and percentage biodegradation of the 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 5 7 9 14	46.86 45.25 45.01 45.91 45.78	43.12 17.48 33.20 39.56 39.36	3.74 27.77 11.81 6.35 6.42	4.1 30.5 13.0 7.0 7.1	4.1 34.7 47.6 54.6 61.7	2 20 27 31 35

¹⁾–Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test item and positive control: 174.7 mg CO₂/2l (ThCO₂test item: 88.8 mg CO₂/2l + ThCO₂sodium acetate: 85.9 mg CO₂/2l)

 

Comparison of biodegradation of the test item in bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	Δ A-B^1)
2 5 7 9 14 19 23 27 29 29 29	2 4 4 4 4 4 4 5 5 5 8	0 0 0 0 0 0 0 0 0 0 1	1 2 2 2 2 2 2 3 3 3 5	2 4 4 4 4 4 4 5 5 5 7

¹⁾–Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Phenol, 1,1-dimethylpropyl derivs. 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) of Phenol, 1,1-dimethylpropyl derivs.

 

The study procedures described in this report were in compliance with 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 ISO 9439, 1999 and ISO 10634, 1995.

 

Phenol, 1,1-dimethylpropyl derivs. was a colourless to pale yellow solid and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l. The Total Organic Carbon (TOC) content of the test substance was determined to be 81%. Based on the TOC content the ThCO₂of the test substance was calculated to be 2.96 mg CO₂/mg. The test substance was tested in duplicate at a concentration of 15 mg/l, corresponding to 12 mg TOC/l.

 

Before use the test substance was heated in a water bath (90°C) to liquefy and homogenize and sub stocks were weighed for several projects. Sub stocks were stored under nitrogen in the dark.

 

The study consisted of six bottles:

- 2 inoculum blanks (no test item),

- 2 test bottles (Phenol, 1,1-dimethylpropyl derivs.),

- 1 positive control (sodium acetate) and

- 1 toxicity control (Phenol, 1,1-dimethylpropyl derivs. plus sodium acetate).

 

On the day of testing weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. An amount of the test substance was weighed form a sub stock. This weighing was performed under light conditions. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. The time between weighing and introduction to the test vessels was maximally 35 minutes, the time required for handling. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and test medium. Test duration was 28 days (last CO₂-measurement on day 29).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Phenol, 1,1-dimethylpropyl derivs. (8% and 1%, based on ThCO₂).

 

In the toxicity control, Phenol, 1,1-dimethylpropyl derivs. 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, Phenol, 1,1-dimethylpropyl derivs. was designated as not readily biodegradable.

Description of key information

Key value determined in a GLP accredited laboratory study using the carbon dioxide (CO₂) evolution test (modified Sturm test) performed in accordance with OECD Guideline 301B and EU Method C4 -C.

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 no significant biodegradation of Phenol, 1,1-dimethylpropyl derivs. (8% and 1%, based on ThCO₂).

In the toxicity control, Phenol, 1,1-dimethylpropyl derivs. was found not to inhibit microbial activity.

In conclusion, Phenol, 1,1-dimethylpropyl derivs. was designated as not readily biodegradable.