4-(1,1-dimethylethyl)cyclohexyl methacrylate

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:

Experimental starting date 24-01-2017 Experimental completion date 03-03-2017

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)

Qualifier:

according to guideline

Guideline:

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

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch/Lot number DAR-16030
Purity: 89.2%

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (19-01-2017). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). 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:

Deionized water
Deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification 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 not added to the river water 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 top 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.

Test procedures
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 containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate. 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.

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle with river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of 4-(1,1-dimethylethyl)cyclohexyl methacrylate, and sodium acetate were calculated from their molecular formulae and molecular weights as follows.

ThOD_NH3(mgO2/mg) = (16 (2C +0.5(H-Cl-3N) + 3S +2.5P + 0.5Na - O))/MW

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles with and without silica gel n-days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Test performance:

The pH of the media was 8.0 at the start of the test. The pH of the media at day 28 was 8.0 (test, control and control with silica gel). 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 1.4 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

28 d

Details on results:

4-(1,1-Dimethylethyl)cyclohexyl methacrylate was biodegraded by 63% at day 28 in the Closed Bottle test. Over 60% biodegradation was not achieved in a period of 14 days immediately following the attainment of 10% biodegradation. The window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Hydrolysis of 4-(1,1-dimethylethyl)cyclohexyl methacrylate however results in the formation of cyclohexanol and methacrylate. The biodegradation kinetics (lag period, growth rate, yield etc.) of the hydrolysis products are not necessarily same. It is thus possible that individual hydrolysis products meet the time window criterion whereas the biodegradability curve of 4-(1,1-dimethylethyl)cyclohexyl methacrylate suggests that the substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion as described for mixtures of chemicals by Richterich and Steber (2001).
4-(1,1-Dimethylethyl)cyclohexyl methacrylate should therefore be classified as readily biodegradable only using the pass level of 60% at day 28.

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 4-(1,1-dimethylethyl)cyclohexyl methacrylate 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.

Parameter:

ThOD

Value:

2.7 g O2/g test mat.

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 4-(1,1-dimethylethyl)cyclohexyl methacrylate is 2.7 g oxygen/g test substance. It was assumed that the ThODs of the proprietary impurities were identical. The ThOD of sodium acetate is 0.8 mg/mg.

Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.7	8.7	8.7	8.7
	8.7	8.7	8.7	8.7
Mean (M)	8.7	8.7	8.7	8.7
7	7.9	7.8	7.9	3.7
	7.9	7.9	7.9	3.9
Mean (M)	7.9	7.9	7.9	3.8
14	7.6	6.6	7.4	3.2
	7.6	6.8	7.5	3.2
Mean (M)	7.6	6.7	7.5	3.2
21	7.3	4.5	7.3
	7.3	4.3	7.3
Mean (M)	7.3	4.4	7.3
28	7.3	3.8	7.2
	7.2	4.0	7.3
Mean (M)	7.3	3.9	7.3

O_csRiver water with nutrients and silica gel.

O_tRiver water with nutrients, test material (2.0 mg/L) and silica gel.

O_cRiver water with nutrients.

O_aRiver water with nutrients and sodium acetate (6.7 mg/L).

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	0.0	4.1	0	76
14	0.9	4.3	17	80
21	2.9		54
28	3.4		63

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

4-(1,1-Dimethylethyl)cyclohexyl methacrylate was biodegraded by 63% at day 28 in the OECD 301 Closed Bottle test. The substance should therefore be classified as readily biodegradable.
4-(1,1-Dimethylethyl)cyclohexyl methacrylate 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.

Executive summary:

In order to assess the biotic degradation of 4-(1,1-dimethylethyl)cyclohexyl methacrylate, 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.

4-(1,1-Dimethylethyl)cyclohexyl methacrylate 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.

4-(1,1-Dimethylethyl)cyclohexyl methacrylate was biodegraded by 63% at day 28 in the Closed Bottle test. Over 60% biodegradation was not achieved in a period of 14 days immediately following the attainment of 10% biodegradation. The window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Hydrolysis of 4-(1,1-dimethylethyl)cyclohexyl methacrylate however results in the formation of cyclohexanol and methacrylate. The biodegradation kinetics (lag period, growth rate, yield etc.) of the hydrolysis products are not necessarily same. It is thus possible that individual hydrolysis products meet the time window criterion whereas the biodegradability curve of 4 -(1,1-dimethylethyl)cyclohexyl methacrylate suggests that the substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion as described for mixtures of chemicals by Richterich and Steber (2001). 4-(1,1-Dimethylethyl)cyclohexyl methacrylate should therefore be classified as readily biodegradable only using the pass level of 60% at day 28.

The test is valid as shown by an endogenous respiration of 1.4 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 substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable