4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

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 start date: 05 July 2017 Experimental completion date: 02 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Physical state/Appearance: very pale yellow liquid
Chemical (IUPAC) Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Storage Conditions: approximately 4 ºC

Oxygen conditions:

aerobic

Inoculum or test system:

other: mixed population of sewage treatment micro-organisms from predominantly domestic sewage

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 05 July 2017 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.1 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately
1-Hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

* CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules
** Whatman GF/A (21 mm diameter) with acetone evaporated off

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the study.
Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels.
An aliquot (24 µL) of the 1070 mg/10 mL test item solvent stock solution was dispensed separately on to 9 filter papers and the solvent allowed to evaporate to dryness for approximately 15 minutes.
The final concentration in the toxicity control vessels was 24.0 mg test item/L plus 34.3 mg reference item/L, equivalent to 40 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper*, 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, plus a filter paper*, to give a final concentration of 20 mg carbon/L, 33 replicate vessels.
c) The test item, on a filter paper*, in inoculated mineral medium to give a final concentration of 20 mg carbon/L, 29 replicate vessels.
d) The test item, on a filter paper*, plus the reference item in inoculated mineral medium to give a final concentration of 40 mg carbon/L to act as a toxicity control,
9 replicate vessels.

A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

Test media a) to d) were inoculated with the prepared inoculum at a final concentration of 100 mL/L.
Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminum crimp caps and incubated in darkness at approximately 20 ºC with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).
* Whatman GF/A (21 mm diameter) with acetone evaporated off

Reference substance:

other: sodium benzoate

Test performance:

The mean TIC in the inoculum control vessels on Day 28 was 0.13 mg/L; equivalent to 1% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Details on results:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
The toxicity control attained 26% biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 showed that the replicate reference item vessels attained 91% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Results with reference substance:

Sodium benzoate attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The substance showed 0% biodegradation in an OECD TG 310 test and is considered to be not readily biodegradable.

Executive summary:

The ready biodegradability of FRET 12-0492 was investigated in a study conducted in accordance with OECD TG 310 (CO2 Headspace Test) and GLP. The concentration tested was 20 mg/l test substance. The test item attained 0% biodegradation after 28 days it was not toxic to the inoculum (26 % biodegradation after 14 days in the toxicity control). The reference substance sodium benzoate attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. 

Description of key information

The ready biodegradability of FRET 12-0492 was investigated in a study conducted in accordance with OECD TG 310 (CO2 Headspace Test) and GLP. The concentration tested was 20 mg/l test substance. The test item attained 0% biodegradation after 28 days it was not toxic to the inoculum (26 % biodegradation after 14 days in the toxicity control). The reference substance sodium benzoate attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater