Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and oxydipropanol

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP ; minor deviations from guideline not considered to impact the overall reliability of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations not considered to have an impact on the reliability of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: van Ginkel and Stroo (1992) Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotox Environ Saf 24 319-327.

Deviations:

not applicable

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301) were introduced: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound)

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: August 2014; signature: October 2014

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): River water was sampled from the Rhine near Heveadorp, The Netherlands. The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The river water was aerated for 7 days before use to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

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:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 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. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively.
Accurate administering of test substance was accomplished by preparing a solid stock of 3.0 mg of 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 top with aluminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel with 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. The controls as appropriate contained 0.2 g silica gel per bottle (Silica gel grade 636, pore size 60A, 35-60 mesh particle size).
- Test temperature: Temperatures were within the prescribed temperature range of 22 to 24°C.
- pH: The pH of the media was 7.1 at the start of the test. The pH of the medium at day 28 was 7.0 (control) and 8.0 (test and control with silica gel)
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing medium with inoculum and silica gel, 10 bottles containing medium with inoculum and silica gel with test substance, 6 bottles containing sodium acetate and medium with inoculum.
- Measuring equipment: See details on analytical methods
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: No.

SAMPLING
- Sampling frequency: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
- Sampling method: analyses of the dissolved oxygen concentration
- Sterility check if applicable: No.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Toxicity control: Not required. Based on absence of inhibition of endogenous respiration of the inoculum detected.

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

60 d

Remarks on result:

other: enhanced biodegradability test: prolonged closed bottle test

Details on results:

The test substance was biodegraded by 61% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 70% at day 60 (enhanced biodegradability testing). This result clearly demonstrates that the test substance is ultimately biodegradable.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 7 was 81%.

- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the reaction mass of the test substance is 2.2 mg/mg (as presented in the full study report). 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 the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.

- Validity of the test: The validity of the test is demonstrated by an endogenous respiration of 1.2 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 89%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

- Biodegradability: The test substance was biodegraded by 61% at day 28 in the Closed Bottle test. The multi-constituent test substance is therefore classified as readily biodegradable. The test substance is degraded by 70% at day 60. The biodegradation percentage of > 60% demonstrates that the test item is ultimately biodegradable.

Table 1. - Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, 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	1.5	4.4	34	81
14	2.2	4.8	50	89
21	2.6		59
28	2.7		61
42	3.0		68
60	3.1		70

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance was biodegraded by 61 % at day 28 in the Closed Bottle test.

Executive summary:

In order to assess the biotic degradation, 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 (OECD TG 301 D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test substance 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. Test substance was biodegraded by 61% at day 28 in the OECD TG 301 Closed Bottle test and hence should be classified as readily biodegradable. In the prolonged Closed Bottle test, the test item was biodegraded by 70% at day 60 (enhanced biodegradability testing). This result clearly demonstrates that the test substance is ultimately biodegradable. The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 89% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period. Under the conditions of this study, this substance should be classified as readily biodegradable and there was evidence of ultimate biodegradability within a time period of 60 days.

Description of key information

Biodegradation: readily biodegradable, 61% (28-days, 14-day window not met), 14-day window not applicable to substances consisting of structurally similar constituents (OECD, 2006), OECD TG 301D, 2016

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In order to assess the biotic degradation, 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 (OECD TG 301 D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test substance 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. Test substance was biodegraded by 61% at day 28 in the OECD TG 301 Closed Bottle test and hence should be classified as readily biodegradable. In the prolonged Closed Bottle test, the test item was biodegraded by 70% at day 60 (enhanced biodegradability testing). This result clearly demonstrates that the test substance is ultimately biodegradable. The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 89% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period. Under the conditions of this study, this substance should be classified as readily biodegradable and there was evidence of ultimate biodegradability within a time period of 60 days.