Methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate

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:

14 Feb 2017 to 17 Apr 2017

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)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Version / remarks:

1994

GLP compliance:

yes (incl. QA statement)

Remarks:

Akzo Nobel ,Environmental Chemistry, Zutphenseweg 10, 7418 AJ Deventer, The Netherlands

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

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-exposed to the test substance.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- pH: 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 (controls) and 7.2 (test).
- Temperature: Temperatures were within the prescribed temperature range of 22 to 24°C.

TEST SYSTEM
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

NUTRIENTS AND STOCKS
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw and the content was mixed vigorously. Subsequently 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

PROCEDURE
Use was made of 10 bottles containing only inocuum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum, silica gel, and test substance, and 6 bottles containing 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. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Reference substance:

acetic acid, sodium salt

Test performance:

VALIDITY
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.

Key result

Parameter:

% degradation (O2 consumption)

Value:

33

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

67

Sampling time:

60 d

Details on results:

For more detail, see 'Any other information on results incl. tables'.

Results with reference substance:

The biodegradation of the reference compound was 83% on day 14.

Table Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD)  

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	1.0	4.1	19	76
14	1.4	4.3	27	80
21	1.6		31
28	1.7		33
42	2.4		46
60	3.5		67

THEORERTICAL OXYGEN DEMAND (ThOD)

The calculated theoretical oxygen demand (ThOD) of Lilial ME Anthranilate (Lilianth BHT) is 2.6 mg/mg. 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 Lilial ME Anthranilate (Lilianth BHT) 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 (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Lilial ME Anthranilate (Lilianth BHT) was biodegraded by 33% at day 28 in the Closed Bottle test. It is not readily biodegradable. In the prolonged Closed Bottle test the test substance is biodegraded by 67% at day 60. A biodegradation in excess of 60% within the 60-day test period allows classification of Lilial ME Anthranilate (Lilianth BHT) as not persistent.

Executive summary:

In order to assess the biotic degradation of Lilial ME Anthranilate (Lilianth BHT), 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.

Lilial ME Anthranilate (Lilianth BHT) 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. Lilial ME Anthranilate (Lilianth BHT) was biodegraded by 33% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 67% at day 60 (enhanced biodegradability testing). It is not readily biodegradable under the testing condition. A biodegradation percentage of >60% within a 60-day time period allows classification of Lilial ME Anthranilate (Lilianth BHT) as not persistent.

.

Description of key information

In order to assess the biotic degradation of Lilial ME Anthranilate (Lilianth BHT), 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.

Lilial ME Anthranilate (Lilianth BHT) 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. Lilial ME Anthranilate (Lilianth BHT) was biodegraded by 33% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 67% at day 60 (enhanced biodegradability testing). It is not readily biodegradable under the testing condition. A biodegradation percentage of >60% within a 60-day time period allows classification of Lilial ME Anthranilate (Lilianth BHT) as not persistent.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater