4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

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:

14.02.1995 - 15.03.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 13 February 1995 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, U. K., which treats predominantly domestic sewage.
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The following test solutions were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final test concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final test concentration of 20 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 30 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The study was carried out at a temperature of 21 °C in darkness.
Approximately 24 hours prior to the start of the study the vessels were filled with 2400 ml of culture medium and 38 ml of inoculum and aerated overnight. On day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and Co2-free air bubbled through the solution at a rate of 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by sparging compressed air through the following series:
i) Three 500 ml Dreschel bottles filled with 350 ml 10 N NaOH
ii) One 500 ml Dreschel bottle filled with 350 ml 0.025 N Ba(OH)2
iii) one empty 500 ml Dreschel bottle to prevent liquid carry-over to the test vessels.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Preliminary investigational work to assess any toxic effect of the test material on sewage sludge micro-organisms following the method described in OECD Guideline No. 209 "Activated Sludge Respiration Inhibition Test" was not performed. Therefore a toxicity control (test material and sodium benzoate) was included in the study to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the study.

Test performance:

Calculation of carbon content:
The theoretical amount present for test material (C28H37NO7) was calculated.
The test material has a purity of >99% and so for a concentration of 20 mg C/l (i.e. a total of 89.1 mg) the total organic carbon present was 60 mg C.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H5.COONa) was calculated.
Thus for a 10 mg/l test concentration (i.e. a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.
Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values.
The percentage degradation from the results of DOC analysis is calculated.
Validation criteria:
The results of the degradation test are considered valid if in the same test the standard material yields >60% degradation by day 14.
The test material may be considered to be readily biodegradable if >60% degradation is attained after 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test material and sodium benzoate) should attain > 25% degradation by day 14 for the test material to be considered as non-inhibitory.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

1 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

2 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

11

Sampling time:

3 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

18

Sampling time:

6 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

24

Sampling time:

8 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

29

Sampling time:

10 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

47

Sampling time:

12 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

48

Sampling time:

14 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

60

Sampling time:

16 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

68

Sampling time:

20 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

71

Sampling time:

22 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

67

Sampling time:

24 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

83

Sampling time:

27 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

86

Sampling time:

28 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

85

Sampling time:

29 d

Details on results:

The test material attained 86% degradation after 28 days. However the "10-day window" validation criterion, whereby >60% degradation must be attained within 10 days of exceeding 10% degradation, was not met and therefore, the test material cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.
The results of the inorganic carbon analysis of both absorber vessels on day 29 confirmed that no significant amounts of CO2 were present in solution in the culture vessels as inorganic carbonate, and that there was no significant carry-over of CO2 into the second absorber.
Analysis of the test media from the test material culture vessels on days 0 and 28 for the dissolved organic carbon (DOC) gave percentage degradation values of 95% and 91& respectively for replicates R1 and R2.
The toxicity control (test material plus sodium benzoate) attained 96 % degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Results with reference substance:

Sodium benzoate attained 101 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The degradation rate in excess of 100% is considered to be due to sampling and/or analytical variation.

Validity criteria fulfilled:

no

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test material attained 86 % degradation after 28 days. However the "10-day window" validation criterion, whereby > 60 % degradation must be attained within 10 days of exceeding 10 % degradation, was not met and therefore, the test material cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.

Executive summary:

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability: CO2 Evolution Test" referenced as Method C.4 -C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

The test material was exposed to sewage sludge micro-organisms at a concentration of 20 mg C/L with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Description of key information

The test material attained 86 % degradation after 28 days. However, the "10-day window" validation criterion, whereby > 60 % degradation must be attained within 10 days of exceeding 10 % degradation, was not met and therefore, the test material cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater