Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

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:

2012-01-27 to 2012-02-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD-Guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

EC Number:

251-073-2

EC Name:

Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Cas Number:

32509-66-3

Molecular formula:

C50H66O8

IUPAC Name:

2-{[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butanoyl]oxy}ethyl 3,3-bis(3-tert-butyl-4-hydroxyphenyl)butanoate

Details on test material:

- Name of test material (as cited in study report): Hostanox O 3 Pills
- Molecular formula: (C25H33O4)2
- Molecular weight: 794 g/mol

:

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 2 - 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 4 days.
10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 3.8 x 10e8 CFU/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

25 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: 21-22 °C
- pH:
pH-Values at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.72 1) 7.83
2) 7.80
Functional Control 7.81 7.89
Test Item 7.73 1) 7.76
2) 7.76
Toxicity Control 7.73 7.97


- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.



SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
Concerning the OECD-guideline 301 the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

No preliminary study

Test performance:

Based on the calculated oxygen demand, the test concentration of 25 mg/L corresponding to an oxygen demand of 61.25 mg O2/L in the vessel was selected.

The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass bottles (test item, toxicity control):

• two for the test item (P1, P2)
• one for the functional control (R1)
• one for the toxicity control (T1)
• two for the inoculum control (C1, C2)

Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:

• one for the test item (PpH)
• one for the toxicity control (TpH)

The test medium, consisting of the required volumes of mineral medium stock solutions, demineralised water and 10 mL/L inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.
The test item was weighed out and was transferred directly into the brown glass bottle with 250 mL test medium.
Accordingly, the test and reference item for the toxicity control were weighed out and transferred directly into the brown glass
bottle.
For the functional control the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The required volumes of mineral medium stock solutions and 10 mL/L inoculum were added, then the flask was filled up with demineralised water. This test solution was placed in the brown glass bottle.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles will be closed with OxiTop measuring heads and the measuring system will be activated.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Details on results:

The biodegradation in % was calculated based on the ThOD of 2.45 mg O2/mg test item.The pass level for ready biodegradation (> 60 % degradation) was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.
In case of the functional control, the adaptation phase changed to degradation phase after
1 day (degradation > 10 %). The pass level > 60 % was reached after 2 days. The biodegradation rate came to a maximum of 97 % on day 26.

Both test item replicates did not reach the 10 % level (beginning of biodegradation). The mean biodegradation on day 28 was 3 %.

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.

BOD5 / COD results

Results with reference substance:

In the toxicity control the biodegradation achieved 48 % after 14 days. After 28 days the biodegradation came to 50 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Any other information on results incl. tables

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control, Test Item Hostanox O 3 Pills and Toxicity Control

		BOD [mg O2/L]
Date	[d]	Inoculum Control	Functional Control 45 mg/L		Test Item 25 mg/L				Toxicity Control 25 mg/L Test Item+ 45 mg/L Reference Item
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
2012-01-28	1	2.1	29.6	27.5	1.4	-0.7	2.8	0.7	28.2	26.1
2012-01-29	2	4.9	52.1	47.2	4.2	-0.7	5.6	0.7	47.9	43.0
2012-01-30	3	5.6	62.0	56.4	5.6	0.0	5.6	0.0	59.2	53.6
2012-01-31	4	6.3	67.6	61.3	7.0	0.7	7.0	0.7	62.0	55.7
2012-02-01	5	5.6	63.4	57.8	4.2	-1.4	5.6	0.0	63.4	57.8
2012-02-02	6	5.6	66.2	60.6	4.2	-1.4	5.6	0.0	66.2	60.6
2012-02-03	7	7.8	67.6	59.9	5.6	-2.2	7.0	-0.8	67.6	59.9
2012-02-04	8	9.9	74.7	64.8	8.5	-1.4	9.9	0.0	70.5	60.6
2012-02-05	9	9.9	76.1	66.2	9.9	0.0	9.9	0.0	71.9	62.0
2012-02-06	10	9.2	73.3	64.1	7.0	-2.2	9.9	0.7	71.9	62.7
2012-02-07	11	7.8	71.9	64.2	4.2	-3.6	8.5	0.8	71.9	64.2
2012-02-08	12	9.2	76.1	67.0	8.5	-0.7	11.3	2.2	73.3	64.2
2012-02-09	13	9.2	76.1	67.0	7.0	-2.2	11.3	2.2	73.3	64.2
2012-02-10	14	9.2	77.5	68.4	8.5	-0.7	11.3	2.2	74.7	65.6
2012-02-11	15	9.2	77.5	68.4	8.5	-0.7	11.3	2.2	74.7	65.6
2012-02-12	16	9.2	80.3	71.2	8.5	-0.7	12.7	3.6	76.1	67.0
2012-02-13	17	9.9	78.9	69.0	8.5	-1.4	12.7	2.8	76.1	66.2
2012-02-14	18	10.6	78.9	68.3	7.0	-3.6	11.3	0.7	76.1	65.5
2012-02-15	19	9.2	77.5	68.4	7.0	-2.2	9.9	0.8	74.7	65.6
2012-02-16	20	10.6	81.7	71.1	8.5	-2.1	12.7	2.1	77.5	66.9
2012-02-17	21	9.2	77.5	68.4	7.0	-2.2	11.3	2.2	76.1	67.0
2012-02-18	22	9.9	80.3	70.5	8.5	-1.4	12.7	2.9	77.5	67.7
2012-02-19	23	9.9	78.9	69.1	7.0	-2.9	11.3	1.5	77.5	67.7
2012-02-20	24	9.9	80.3	70.5	8.5	-1.4	12.7	2.9	77.5	67.7
2012-02-21	25	9.2	78.9	69.8	7.0	-2.2	11.3	2.2	77.5	68.4
2012-02-22	26	9.9	83.1	73.3	8.5	-1.4	12.7	2.9	77.5	67.7
2012-02-23	27	11.3	84.5	73.2	9.9	-1.4	14.1	2.8	78.9	67.6
2012-02-24	28	11.3	84.5	73.2	9.9	-1.4	14.1	2.8	78.9	67.6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item Hostanox O 3 Pills was determined with non adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F. The test item concentration selected as appropriate was 25 mg/L, corresponding to a ThOD of 61.25 mg O₂/L per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 11.3 mg O₂/L on day 28.

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 2 days. The biodegradation rate reached a maximum of 97 % degradation on day 26.

In the toxicity control containing both test and reference item 48 % degradation occurred within 14 days. After 28 days the biodegradation came to 50 %. The degradation of the reference item was not inhibited by the test item.

The biodegradation of the test item is shown in Table 1 and graphically presented in Figure 1 in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10 % level (beginning of biodegradation). The mean biodegradation on day 28 was 3 %.

The test item is classified as

not readily biodegradable

in the 10-d-window and after 28 days.

Biodegradation of the Test Item Hostanox O 3 Pills in Comparison to the
Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 25 mg/L	1	0	0	0	0
	2	0	4	4	5
Functional Control 45 mg/L		80	91	91	97
Toxicity Control 25 mg/L Test Item + 45 mg/L Reference Item		44	48	49	50