2,4-dimethylcyclohex-3-ene-1-methyl acetate

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:

27 September 2016 - 28 November 2016

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (22-09-2016) 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. The inoculum was not pre-exposed to the test substance.

Duration of test (contact time):

28 d

Initial conc.:

1 mg/L

Based on:

test mat.

Remarks:

coated on silica gel

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

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

Nutrients, stocks and administration:
The nutrient medium of the Closed Bottle test contained per liter of deionized water; 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 top and the content was mixed vigorously. Subsequently 0.10 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 1.0 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 procedure:
Use was made of 10 bottles containing only inoculum (inoculum blank), 10 bottles containing inoculum and silica gel (silica gel control), 10 bottles containing inocolum, silica gel and test substance, and 6 bottles with inoculum and sodium acetate (positive control). The concentrations of the test substance, and sodium acetate in the bottles were 1.0 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.

Test conditions:
The pH of the media were 7.3 at the start of the test. The pH of the media at day 28 were also 7.3 (both test and controls). Temperatures were within the prescribed temperature range of 22 to 24°C.

Reference substance:

acetic acid, sodium salt

Remarks:

purity > 99%

Test performance:

The test is valid as shown by an endogenous respiration <1.5 mg/L (i.e. 0.9 mg/L) at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Oxygen concentrations were >0.5 mg/L in all bottles during the test period.

Key result

Parameter:

% degradation (O2 consumption)

Value:

28

Sampling time:

28 d

Details on results:

- The substance is biodegraded by 28% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test the test substance was biodegraded by 48% at day 60. The test item therefore did not fulfill the criteria for readily biodegradation (>60%) after 28 days of incubation. Hence the substance should be classified as non readily biodegradable.

- The calculated theoretical oxygen demand (ThOD) of the substance is 2.5 g/g. The ThOD of sodium acetate is 0.8 g/g

- No toxicity control was used in this test since possible toxicity of the test substance to microorganisms degrading acetate is not considered 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.

- Functioning of the test system was checked by testing the reference item (sodium acetate), which showed a normal biodegradation curve.

Results with reference substance:

81% after 14 days.

Validity criteria fulfilled:

yes

Remarks:

Endogenous respiration was 0.9 mg/L at day 28, the differences of the replicate values at day 28 were < 20%, the reference item was degraded by 81% of its ThOD after 14 days, oxygen concentrations were >0.5 mg/L in all bottles throughout the test

Interpretation of results:

not readily biodegradable

Conclusions:

The substance is biodegraded by 28% at day 28 in the Closed Bottle test and should therefore be classified as not readily biodegradable under the test conditions in this study.

Executive summary:

In order to assess the biodegradation of the test substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. In this study activated sludge (2 mg/L) from a domestic wastewater treatment plant was exposed to 1 mg/L of the substance for 28 days. The test substance was biodegraded by 28% at day 28 in the Closed Bottle test and should therefore be classified as not readily biodegradable. The acceptability criteria were met and therefore the test was considered to be valid.

Description of key information

In order to assess the biodegradation of the test substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. In this study activated sludge (2 mg/L) from a domestic wastewater treatment plant was exposed to 1 mg/L of the substance for 28 days. The test substance was biodegraded by 28% at day 28 in the Closed Bottle test and should therefore be classified as not readily biodegradable. The acceptability criteria were met and therefore the test was considered to be valid.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed