1-Amino-4-oxocyclohexanecarboxylic acid ethylene ketal

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May - 04 Jun 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Publication No. L 142/496, May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-Amino-4-oxocyclohexanecarboxylic acid ethylene ketal

EC Number:

686-994-2

Cas Number:

54621-18-0

Molecular formula:

C9H15NO4

IUPAC Name:

1-Amino-4-oxocyclohexanecarboxylic acid ethylene ketal

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Storage conditions: 22 °C ± 1 °C, Darkness
- Storage length: overnight
- Preparation of inoculum for exposure: 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 overnight. This suspension was used for the experiment.
- Concentration of sludge: 3.5 g dry material per litre
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

195.5 mg/L

Based on:

ThOD

Remarks:

(ThODNO3)

Initial test substance concentration 2

Initial conc.:

162.9 mg/L

Based on:

ThOD

Remarks:

(ThODNH4)

Initial test substance concentration 3

Initial conc.:

102.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

The change of pressure in the test flasks was measured by means of a manometric method (BSB-Sensomat-System©).

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water
- Test temperature: 22 °C ± 1 °C
- pH: 7.5 (measured at the start of the test), 7.3 to 7.5 (measured at the end of the test)
- Aeration of dilution water: no
- Suspended solids concentration: 3.5 g dry material per litre
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BSB-Sensomat-System©
- Number of culture flasks/concentration: 2 per test item treatment and inoculum control, 1 per procedure control, abiotic control and toxicity control
- Method used to create aerobic conditions: aerated of the prepared sludge suspension overnight before test start
- Measuring equipment: The change of pressure in the test flasks was measured by means of a manometric method (BSB-Sensomat-System©).
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB-Sensomat-System©. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: contaning activated sludge and test water (2 replicates)
- Abiotic sterile control: containing test item, CuSO4 solution and test water (1 replicate)
- Toxicity control: containing test item, reference item, activated sludge and test water (1 replicate)

STATISTICAL METHODS: No

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

none

Test performance:

Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
Incubation: The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks.
Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

% Degradation

% Degradation 1

Key result

Parameter:

other: ThOD (NO3)

Value:

ca. 4

Sampling time:

28 d

% Degradation 2

Key result

Parameter:

other: ThOD (NH4)

Value:

ca. 5

Sampling time:

28 d

Details on results:

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. 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 test item never reached 10% degradation or more during the incubation time. The degradationat test end at day 28 was 6% based on ThODNH4 and 3% based on ThODNO3.

BOD5 / COD results

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 82% after 14 days and to 93% after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Any other information on results incl. tables

Results:

In the toxicity control containing both, the test item and the reference item sodium benzoate, 38% (ThOD_NH4) biodegradation was noted within 14 days and 41% (ThOD_NH4) biodegradation after 28 days of incubation (34% and 38% based on ThOD_NO3).

Time	% biodegradation	% biodegradation	% biodegradation	% biodegradation
days	test item 1	test item 2	sodium benzoate	toxicity control
1	0	0	24	12
2	0	0	38	18
3	1	0	46	23
4	0	0	53	25
5	0	0	59	26
6	0	0	65	29
7	0	0	68	29
8	0	0	71	30
9	0	3	74	32
10	0	3	74	32
11	0	3	76	33
12	0	3	76	33
13	0	3	79	33
14	3	3	82	34
15	3	3	82	34
16	3	3	85	36
17	3	3	85	36
18	3	3	88	37
19	3	3	88	37
20	3	3	88	37
21	3	3	91	37
22	3	3	91	37
23	3	3	91	37
24	3	5	94	38
25	3	5	94	38
26	1	4	93	38
27	1	4	93	38
28	4	4	93	38

 

Validity Criteria: 

Validity criteria	value required by guideline	obtained value	criteria fulfilled
oxygen demand of the inoculum control	60 mg O2/L within 28 days	7.5 mg O2/L	yes
pH-value of the test item flasks at the end	pH 6.0 to 8.5	7.4	yes
degradation of reference item sodium benzoate	> 60% within 14 days	82%	yes
difference of duplicate values for the degradation of the test item at test end	< 20%	0%	yes
toxicity test, containing both the test item and a reference item	< 25% biodegradation (based on ThODNH4) occurred within 14 days	The biodegradation was 38 % at day 14	the test item was not inhibitory.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see above

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of the test item did not reach 60% within the 10-day window or after 28 days.
Therefore, the test item is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.

Executive summary:

Test Design: The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 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.

Endpoints: Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.

Test Item Loading Rate (initial concentration in medium C₀): 102.5 mg/L corresponding to an oxygen demand of about 162.9 mg/L (ThOD_NH4) and 195.5 mg/L (ThOD_NO3)

Reference Item: Sodium benzoate, 102.0 mg/L corresponding to an oxygen demand of about 170.0 mg/L (ThOD_NH4)

Test Conditions: 22 °C ± 1 °C, darkness

Results:

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThOD_NO3) was considered for the evaluation of the results. 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 test item never reached 10% degradation or more during the incubation time. The degradation at test end at day 28 was 5% based on ThOD_NH4 and 4% based on ThOD_NO3.

Conclusion:  The test item is considered to be not readily biodegradable based on ThOD_NH4 and ThOD_NO3.