Ethylene dibenzoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS No.: 94-49-5
Molecular formula: C16H14O4
Solubility in water: insoluble
Supplier of Test Item :. Shiva Pharmachem Ltd.
Lot number: 100000005795
Appearance: White flakes
Purity: 98.99%

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh sample of activated sludge was collected from a sewage treatment plant as recommended by the Guidelines for the Testing of Chemicals, MEP, PR. China and the OECD Guidelines for the Testing of Chemicals. A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 3 days before the test exposure was started. It is a well functioning treatment plant receiving predominantly domestic sewage.

Duration of test (contact time):

28 d

Initial conc.:

25.14 mg/L

Based on:

test mat.

Initial conc.:

52.04 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The inoculum was pretreated as follows: coarse particles were removed by filtration through a fine sieve and the sample was settled. After the supernatant was discarded, the sludge was washed twice with mineral medium and the concentrated sludge was suspended in mineral
medium. Keep the suspended sludge aerobic. 3 replicates of 10 mL suspended sludge was weighed and dried at 105°C for 2 hours and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 10.22 g/L as suspended solids (SS) and the suspended sludge was prepared with mineral medium to yield a concentration of 4.0 g/L as SS. The suspended sludge was kept aerobic at 21.2 ~ 23.3 °C. 011 the day of the test beginning, 5 replicates o f 10 ml sludge were weighed and dried at 105°C for 1.5 hours and reweighed. The mean determined concentration was 3620 mg/L as SS. From this result, the added amount of wet sludge was calculated.

Preparation of Inoculated Mineral Medium:
About 900 mL mineral medium was added to l L flask, followed by the addition of 8.29 mL of the prepared activated sludge and the mixture was made up to volume to give a suspended solid concentration of 30 mg/L. The 1 L inoculated mineral medium was brought into each test vessel.

Test Item Test Vessels:
The method of test item addition was as followed: 0.2514 g of test item was weighed and dissolved with 10.00 mL of dichloromethane to obtain a stock solution of test item at the concentration of 25.14 mg/mL. 1.000 mL of the stock solution was applied into the dry test vessel and the vessel was rotated to allow dichloromethane to evaporate and leave a thin coating of test item on the inner surface. Once a thin coating was achieved on the inner surface, 1 L of inoculated mineral medium was brought into the test vessel. The final concentration of test item was 25.14 mg/L, equivalent to 52.04 mg/L ThOD_NH3 in the test suspension. Test item test vessels were prepared in duplicate as test suspension.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

89.7

Sampling time:

28 d

Details on results:

Based on the percentage biodegradation expressed as average, the percentage biodegradation of test item after 2 days averaged 25.2% (2 replicate), which was the initial day on which 10% biodegradation was exceeded, and when the 10—day window began. The percentage biodegradation in activated sludge were 79.0% (2 replicate) within 10-day window (after 12 days), and 89.7% after 28 days.

In the Toxicity Control (TC) test mixture, 87.1% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test.

Results with reference substance:

Biodegradation of the reference substance (Sodium benzoate) attained 66.5% after Day 4, 89.1% after Day 14 and 94.0% after Day 28.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Ethylene glycol dibenzoate is readily biodegradable.

Executive summary:

The ready biodegradability test of Ethylene glycol dibenzoate was conducted with unacclimatised sewage micro-organism by measuring oxygen consumption over the 28-day test period.
The test item was applied into test vessels by a dichloromethane stock solution of test item and dichloromethane was allowed to evaporate and leave a thin coating of test
item on the inner surface, the theoretical concentration was 25.14 mg/L (equivalent to 52.04 mg/L as Theoretical Oxygen Demand or “ThOD_NH3”) (2 replicate). The test concentration of sodium benzoate, used as reference substance in the Procedure Control, was 31.44 mg/L (equivalent to 52.50 mg/L as ThOD). In the Toxicity Control
(TC) containing both test item and reference substance, the test item was added to the test vessel at a level of 25.14 mg/L (equivalent to 52.04 mg/L as ThOD_NH3) and the reference substance was added at a level of 31.63 mg/L of sodium benzoate (equivalent to 52.82 mg/L as ThOD), the concentration expressed as total ThOD was 104.86 mg/L.

Based on the percentage biodegradation expressed as average, the percentage biodegradation of test item afler 2 days averaged 25.2% (2 replicate), which was the
initial day on which 10% biodegradation was exceeded, and when the 10—day window began. The percentage biodegradation in activated sludge were 79.0% (2 replicate)
within 10-day window (after 12 days), and 89.7% after 28 days.
Biodegradation of the reference substance (Sodium benzoate) attained 66.5% after Day 4, 89.1% after Day 1 4 and 94.0% after Day 28.
In the Toxicity Control (TC) test mixture, 87.1% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test
item can be assumed to be not inhibitory under the conditions of this test.

Based on the results, the percentage biodegradation within 10-day window exceeded pass level of 60% ThOD, the test item, Ethylene glycol dibenzoate met the criteria for “ready biodegradability” under the described test conditions.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch/Lot number: 10000005282
Purity: 99.4%
Appearance: powder
Solubility in water: insoluble in water
Storage: at ambient temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Secondary activated sludge (09-02-2017) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L (van Ginkel and Stroo, 1992). The inoculum was not pre-adapted to the test substance.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles with test substance and inoculum, 10 bottles treated with DCM (added and evaporated) with inoculum, 10 bottles only containing inoculum and 6 bottles with sodium acetate and inoculum.
The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

81

Sampling time:

28 d

Details on results:

Ethylene glycol dibenzoate was biodegraded by 81% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation at day 1. Ethylene
glycol dibenzoate therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80.

Theoretical oxygen demand (ThOD)

The calculated theoretical oxygen demand (ThOD) of ethylene glycol dibenzoate is 2.1 g oxygen/g test substance. The ThOD of sodium acetate is 0.8 mg/mg.

Toxicity

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of ethylene glycol dibenzoate to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions

The pH of the media was 7.3 at the start of the test. The pH of the media at day 28 was 7.3 (test, control (DCM added and evaporated), standard control). Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%.

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	3.1	4.1	74	76
14	3.3	4.3	79	80
21	3.3		79
28	3.4		81

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Ethylene glycol dibenzoate should be classified as readily biodegradable.

Executive summary:

In order to assess the biotic degradation of ethylene glycol dibenzoate, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

Ethylene glycol dibenzoate did not cause a reduction in the endogenous respiration at day 7.

The test substance is therefore considered to be non-inhibitory to the inoculum. Ethylene glycol dibenzoate was biodegraded by 81% at day 28 in the OECD 301 Closed Bottle test.

Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation thereby fulfilling the time window criterion for ready biodegradable substances. The substance should therefore be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 0.8 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 80% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

Ethylene glycol dibenzoate did not cause a reduction in the endogenous respiration at day 7.

The test substance is therefore considered to be non-inhibitory to the inoculum. Ethylene glycol dibenzoate was biodegraded by 81% at day 28 in the OECD 301 Closed Bottle test.

Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation thereby fulfilling the time window criterion for ready biodegradable substances. The substance should therefore be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 0.8 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 80% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

This result is supported by an OECD 301F performed in 2018 in China where 89.7% biodegradation was found after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable