Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

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:

29 April 2011 - 01 August 2011

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)

Version / remarks:

(2006)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on April 29, 2011. The Cambridge facility treats predominantly residential wastes.
- Storage conditions: The sludge was sieved using a 2-mm screen and then aerated at test temperature until its next adjustment.
- Pre-treatment: The activated sludge was diluted in test medium to approximately 30 mg total suspended solids/L and then aerated with CO2-free air for approximately one week until its final adjustment on Day 0 of the initial test (pre-conditioning).
- Preparation of inoculum for exposure: Dilution of the pre-conditioned activated sludge in test medium to approximately 4 mg total suspended solids/L followed by aeration for approximately 30 minutes

Duration of test (contact time):

28 d

Initial conc.:

ca. 20 other: mg C/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST DURATION
In addition to the standard OECD 310 test, an enhanced biodegradation screening test, also based upon OECD 310 Guidelines, was performed. The enhancements included low-level pre-adaptation, by utilization of inoculum pre-exposed to the test substance concurrently with the conduct of the first test and by extending the test duration to 60 days.

TEST CONDITIONS
- Composition of medium: 1 mL of 2.75% (w/w) CaCl2 solution in water, 1 mL of 0.025% (w/w) ferric chloride solution in water, 1 mL of 2.25% magnesium sulfate solution in water and 10 mL mixed phosphate buffer (pH 7.4) were made up to 1 L with high quality water (e.g. Nanopure)
- Test temperature: 19.6-21.9°C
- pH: actual value not reported; assumed 7.4 at the start of the study (pH of the mixed phosphate buffer according to OECD310 mineral salts medium)
- pH adjusted: not reported
- Aeration of dilution water: no
- Suspended solids concentration: 4 mg/L in final test solutions
- Continuous darkness: assumed, but not reported

TEST SYSTEM
- Test vessel: glass serum bottles with a nominal volume of 160 mL, sealed with Teflon septa and crimp caps
- Fill volume: 107 mL inoculated test medium was added to the 160 mL bottle, establishing the required headspace to liquid ratio of approximately 1:2
- Number of culture flasks/concentration: 3 replicates per blank control, treatment (1 test concentration) and activity control (2 reference substances)
- Method used to create aerobic conditions: headspace (1-week long preconditioning of the inoculum)
- Measuring equipment: IC analysis, not further specified
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Sampling frequency: every 7 days
- Sampling method: conversion of CO2 to carbonate through addition of 1 mL of 7 M NaOH to the test solutions (assumed via septum), shaking for 1 hour followed by a settling period prior to sample removal for IC analysis
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 bottle
- Abiotic sterile control: none
- Toxicity control: none

STATISTICAL METHODS: only means and corresponding standard deviations were calculated; no further statistical analysis of the data

Reference substance:

other: 1-octanol

Remarks:

(73.83% carbon content, as determined by elemental analysis)

Reference substance:

other: rapeseed oil

Remarks:

(77.26% carbon content, as determined by elemental analysis)

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

32.8

Sampling time:

28 d

Remarks on result:

other: approximately 4 mg suspended solids/L

Parameter:

% degradation (inorg. C analysis)

Value:

29.9

Sampling time:

60 d

Remarks on result:

other: approximately 0.8 mg suspended solids/L

Details on results:

The viability of the inoculum and validity of the test were supported by the results of the reference substances, rapeseed oil and 1-octanol, from which an average of 77.3% and 77.7% of theoretical IC were evolved respectively after 28 days in the standard test.

After 28 days in the second (enhanced) test, averages of 65.7% and 61.6% of theoretical IC were evolved, respectively, and averages of 75.6% and 59.6% were evolved after 60 days. The average cumulative percent biodegradation for Galbanone at the end of the 60-day second test was 29.9%.

Due to the fact that the inoculated test medium for the enhanced test was prepared by combining 200 milliliters of the pre-exposed inoculated test medium, prepared at the start of the initial test and incubated for its duration, per 800 milliliters of freshly prepared test medium, the suspended solids concentration in the enhanced test was 0.8 mg/L, thus much lower than in the standard biodegradation headspace test (4 mg/L).

The average cumulative % biodegradation in the enhanced test (after 60 days) was not markedly different form the % biodegradation in the standard test (after 28 days).

The validity criteria of the standard 28-day test are fulfilled:

1. the mean percentage degradation of the reference substance was >60% by the 14th day of incubation (i.e. 79.9% for rapeseed oil and 75.7% for 1 -octanol)

2. the mean amount of IC present in the blank controls at the end of the test was <3mg C/L (i.e. 1.0 mg C/L).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The substance was found to be not readily biodegradable under the conditions of the OECD 310 headspace test.

Executive summary:

The ready biodegradability of the substance was investigated in a study conducted in accordance with OECD TG 310 and GLP. The concentration tested was 20 mg C/L test substance, with a suspended solids concentration of 4 mg/L The test substance biodegrades for 32.8% in the standard 28d test. No toxicity control was included. However, since no effects were observed at a loading rate of 46 mg/L in the OECD 209 study, it can be assumed that at the test concentration of 20 mg C/L the test substance was not toxic to the inoculum.

In addition to the standard test, an enhanced test was performed, following the same test set-up and procedures according to OECD 310 as the standard test, but using low-level pre-adapted inoculum (at 0.8 mg suspended solids/L) over a duration of 60 days.The average cumulative % biodegradation in the enhanced test (29.9% after 60 days) was not markedly different from the % biodegradation in the standard test.

Description of key information

It is concluded that the substance is not readily biodegradable under the conditions of the OECD 310 headspace test.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the substance was investigated in a study conducted in accordance with OECD TG 310 and GLP. The concentration tested was 20 mg C/L test substance, with a suspended solids concentration of 4 mg/L. The test substance biodegrades for 32.8% in the standard 28d test. No toxicity control was included. However, since no effects were observed at a loading rate of 46 mg/L in the OECD 209 study, it can be assumed that at the test concentration of 20 mg C/L the test substance was not toxic to the inoculum. In addition to the standard test, an enhanced test was performed, following the same test set-up and procedures according to OECD 310 as the standard test, but using low-level pre-adapted inoculum (at 0.8 mg suspended solids/L) over a duration of 60 days. The average cumulative % biodegradation in the enhanced test (29.9% after 60 days) was not markedly different from the % biodegradation in the standard test.

The conclusion as not readily biodegradable is supported by an additional available supporting study, also performed according to OECD 310 and in compliance with GLP.