Isopropyl acetoacetate

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:

2020-04-28 to 2020-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: sewage treatment plant Rossdorf, Germany

- Preparation of sludge: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. 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 aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

102.3 mg/L

Based on:

test mat.

Remarks:

corresponding to an oxygen demand of about 193.0 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water
- Test temperature: 22 °C ± 1 °C
- pH: 7.4 (measured at the start of the test); 7.4 to 7.6 (measured at the end of the test)
- Aeration of dilution water: no
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD Sensor system
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head
- Test performed in open system: no

SAMPLING
- Sampling frequency: every day of the exposure period

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

28 d

Remarks on result:

other: Based on ThODNH4

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 80% after 28 days of incubation.

Validity Criteria of the Study

Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O₂/L and thus not greater than 60 mg O₂/L within 28 days as required by the test guideline.

pH-Value: The pH-value of the test item flasks at the end of the test were 7.5 and 7.4 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (> 60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation

Test Item: The difference of duplicate values for the degradation of the test item at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at days 14 and 28 was 1%. The validity criterion was fulfilled.

Toxicity Control: If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on ThOD_NH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 64% at day 14; the test item was not inhibitory.

 

Biodegradation of Test Item

The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation. The 10-day windows began on day 4 after application, the mean value was calculated to be 18% biodegradation (ThOD_NH4). Therefore, the end of the 10-day window was day 14. The mean biodegradation percentage based on ThODNH4 at the end of the 10-day window was 60%; the criterion of the 10 day window was passed. The mean biodegradation percentage at the end of the 28-day exposure period was 69% (ThOD_NH4).

 

Biodegradation of Reference Item

The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 80% after 28 days of incubation.

 

Biodegradation in the Toxicity Control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 64% (ThOD_NH4) biodegradation was noted within 14 days and 74% (ThOD_NH4) biodegradation after 28 days of incubation.

 

Abiotic Control

The oxygen demand in the abiotic control was 0 mg/L during the test duration.

 

Table 1: Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

Time (Days)	Flask no.
	Flask 1	Flask 2	Flask 3	Flask 4	Flask 5	Flask 6	Flask 7
1	5	0	0	0	55	0	55
2	10	10	5	5	80	0	80
3	25	20	5	5	95	0	95
4	45	35	5	5	110	0	110
5	50	50	10	5	115	0	125
6	60	60	10	5	125	0	135
7	70	65	10	10	135	0	160
8	80	75	10	10	135	0	170
9	95	85	10	10	140	0	185
10	110	95	15	10	145	0	195
11	120	105	15	15	145	0	210
12	125	115	15	15	145	0	220
13	130	125	15	15	150	0	235
14	130	130	15	15	150	0	250
15	135	130	20	15	155	0	265
16	135	135	20	20	155	0	280
17	140	135	20	20	155	0	285
18	140	135	20	20	155	0	290
19	145	140	20	20	155	0	290
20	145	145	25	20	160	0	290
21	145	145	25	20	160	0	290
22	150	150	25	20	160	0	290
23	150	150	25	20	160	0	290
24	150	155	25	20	160	0	290
25	155	155	25	20	160	0	290
26	155	155	25	20	160	0	290
27	155	155	25	20	160	0	290
28	155	155	25	20	160	0	295

Flasks 1 and 2: Test item; Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control); Flask 6: abiotic control

Flask 7: toxicity control

 

Table 2: Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time (Days)	Percentage Biodegradation
	Test item^1		Reference time^2	Toxicity Control^1,2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	3	0	32	15
2	3	3	44	20
3	10	8	53	24
4	21	15	61	29
5	22	22	63	32
6	27	27	69	35
7	31	28	73	41
8	36	33	73	44
9	44	39	76	48
10	51	43	77	50
11	55	46	76	53
12	57	52	76	56
13	60	57	79	60
14	60	59	79	64
15	61	58	80	67
16	60	59	79	71
17	63	59	79	72
18	63	59	79	73
19	65	63	79	73
20	64	63	80	73
21	64	66	80	73
22	66	66	80	73
23	66	68	80	73
24	66	68	80	73
25	69	68	80	73
26	69	68	80	73
27	69	68	80	73
28	69	68	80	74

₁ ThOD_NH4 of the test item: 1.887 mg O2/mg test item

₂ ThOD_NH4 of sodium benzoate: 1.666 mg O2/mg reference item

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions according to OECD 301 F, the test item is considered to be readily biodegradable based on ThODNH4.

Executive summary:

The ready biodegradability of test item was investigated in a Manometric Respirometry test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms 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. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 102.3 mg/L, corresponding to an oxygen demand of about 193.0 mg/L (ThOD_NH4) while the reference item sodium benzoate was tested at a concentration of 102.9 mg/L corresponding to an oxygen demand of about 171.4 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 79 % after 14 days and to 80 % 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, 64 % (ThOD_NH4) biodegradation was noted within 14 days and 74 % (ThOD_NH4) biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25 % within 14 days. A mean biodegradation of 1 0% or more of the test item was reached at day 4 (ThOD_NH4). At the end of the 10-day window at day 14, the mean degradation of the test item was 60 % (ThOD_NH4) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 69 % (ThOD_NH4). Therefore, the test item is considered to be readily biodegradable based on ThOD_NH4.

Description of key information

Under the test conditions according to OECD 301 F, the test item is considered to be readily biodegradable based on ThODNH4 (Reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of test item was investigated in a Manometric Respirometry test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms 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. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 102.3 mg/L, corresponding to an oxygen demand of about 193.0 mg/L (ThOD_NH4) while the reference item sodium benzoate was tested at a concentration of 102.9 mg/L corresponding to an oxygen demand of about 171.4 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 79 % after 14 days and to 80 % 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, 64 % (ThOD_NH4) biodegradation was noted within 14 days and 74 % (ThOD_NH4) biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25 % within 14 days. A mean biodegradation of 10 % or more of the test item was reached at day 4 (ThOD_NH4). At the end of the 10-day window at day 14, the mean degradation of the test item was 60 % (ThOD_NH4) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 69 % (ThOD_NH4). Therefore, the test item is considered to be readily biodegradable based on ThOD_NH4.