Triethyl O-acetylcitrate

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:

2010-04-30 - 2010-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): micro-organisms from a domestic wastewater treatment plant (sewage works of Darmstadt, Germany).
- Preparation of inoculum for exposure:
- Pretreatment: Sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. The sediment was re-suspended in test water and aerated overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated ali-quots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

102 mg/L

Based on:

test mat.

Initial conc.:

159 mg/L

Based on:

other: ThOD(NH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with deionised water to 1000 mL volume
b) 22.5 g MgSO4 x 7H2O filled up with deionised water to 1000 mL volume
c) 36.4 g CaCl2 x 2H2O filled up with deionised water to 1000 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of concentrated HCl per litre was added.
10 mL of stock solution a) and 1 mL of the stock solutions b) to d) were combined and filled up to a final volume of 1000 mL with deionised water.
- Test temperature: 22°C
- pH: 7.5 - 7.6 (measured at the start of the test); 7.1-7.8 (measured at the end of the test)
- pH adjusted: no
- Continuous darkness: yes
- Other: Surrounding Type: Climatised room

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: 7 flasks/ stock suspension of 1.5 g/L on dry matter base (final concentration: 26 g/L)
- Measuring equipment: BSB Sensomat system, Aqualytic Dortmund
- Test performed in closed vessels due to significant volatility of test substance: The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 5 mL activated sludge and 239 mL test water in each of 2 flasks (#3 & #4)
- Abiotic control: 25.3 mg test item, 5 mL activated sludge and 239 mL test water poisoned with HgCl2 (stock solution of 48.84 mg/mL) in flask #6
- Toxicity control: 25 mg test item, 24.9 mg reference item, 5 mL activated sludge and 239 mL test water in flask #7
- Other: Procedure control: 25.0 mg reference item, 5 mL activated sludge and 239 mL test water in flask #5

Reference substance:

other: sodium benzoate

Preliminary study:

No information about a preliminary study is available.

Test performance:

The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the micro organisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
The degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.

Parameter:

% degradation (O2 consumption)

Value:

75

Sampling time:

28 d

Remarks on result:

other: Flask #1

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Flask #2

Details on results:

Biodegradation of the Toxicity Control: In the toxicity control containing both, the test item and the reference item sodium benzoate, 56% biodegradation was noted within 14 days and 64% biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge micro organisms.

Parameter:

BOD5

Value:

150 other: mg O2/L

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 88% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Percentage Biodegradation of Test Item, of Reference substance and of the Toxicity Control based on ThOD_{N H4}

Time [days]	Flask #1- test substance [%]	#2 - test substance [%]	#3 - reference substance [%]	#4 - toxicity control [%]
1	3	3	32	17
10	44	44	82	50
20	66	67	88	61
28	75	76	88	64

Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks During the Test Period of 28 Days

Time [days]	Flask #1	#2	#3	#4	#5	#6	#7
1	5	5	0	0	55	0	55
10	80	80	10	10	150	0	175
20	125	125	20	20	170	0	220
28	150	150	30	30	180	0	240

Validity criteria fulfilled:

yes

Remarks:

All validity criteria of the test method were met.

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Within 28 days, a mean degradation of 76% (ThODNH4) was determined for the test substance; the 10 day window criterion was not passed. Therefore, the test substance is considered to be readily, but failing the 10-day window.

Executive summary:

The biodegradability of the substance 'triethyl O-acetylcitrate' was investigated according to EU method C.4 -D and OECD guideline 301 F (Feil, N., 2010). The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). A measured volume of inoculated mineral medium, containing the test item in a concentration of 102 mg/L, which corresponds to an oxygen demand of about 159 mg/L (ThOD_NH4), was stirred in a closed flask at a temperature of 22 °C for up to 28 days. Aerobic activated sludge, micro organisms from a domestic waste water treatment plant was taken as inoculum. The concentration of the inoculum was 26 mg/L. Analytical grade salts were added to deionised water to prepare the stock solutions. Sodium benzoate (purity of 100 %) was used as reference compound. Also a toxicity control (test item and reference compound mixed) was run in parallel, to ensure, that the chosen test concentration was not inhibitory to microorganims. The degradation rate of the test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. As the test item contains no nitrogen, the evaluation of biodegradation has to be based ThOD_NH4. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.

The mean biodegradation after 28 days of the test substance was 76% (ThOD_NH4); the 10 day window criterion was not passed and therefore it is considered to be readily biodegradable, but failing the 10 -day window. The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 88% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 56% biodegradation was noted within 14 days and 64% biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge micro organisms and all validity criteria of the test method were met.

Description of key information

The test substance can be considered to be  "readily biodegradable"; the 10 day window criterion was not passed.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

The biodegradability of the substance 'triethyl O-acetylcitrate' was investigated according to EU method C.4 -D and OECD guideline 301 F (Feil, N., 2010). The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). A measured volume of inoculated mineral medium, containing the test item in a concentration of 102 mg/L, which corresponds to an oxygen demand of about 159 mg/L (ThOD_NH4), was stirred in a closed flask at a temperature of 22 °C for up to 28 days. Aerobic activated sludge, micro organisms from a domestic waste water treatment plant was taken as inoculum. The concentration of the inoculum was 26 mg/L. Analytical grade salts were added to deionised water to prepare the stock solutions. Sodium benzoate (purity of 100 %) was used as reference compound. Also a toxicity control (test item and reference compound mixed) was run in parallel, to ensure, that the chosen test concentration was not inhibitory to microorganims. The degradation rate of the test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. As the test item contains no nitrogen, the evaluation of biodegradation has to be based ThOD_NH4. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.

The mean biodegradation after 28 days of the test substance was 76% (ThOD_NH4); the 10 day window criterion was not passed and therefore it is considered to be readily biodegradable, but failing the 10 -day window. The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 88% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 56% biodegradation was noted within 14 days and 64% biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge micro organisms and all validity criteria of the test method were met.