Potassium (2S)-4-carboxy-2-(tetradecanoylamino)butanoate

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:

18 January 2012 to 15 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Sewage plant for domestic sewage in Veszprém, Hungary.
- Preparation: The sludge was washed by centrifugation filtered through cotton wool and aerated until use.
- Based on the determined dry weight content, the washed sludge suspension contained 4 g dry material per litre

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
- Stock solutions: In deionised water analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125 g; K2HPO4 5.4375g; Na2HPO4 x 12H2O 16.795g; NH4Cl 0.125 g; Deionised water ad 250 mL
b) Solution: MgSO4 x 7 H2O 5.625 g; Deionised water ad 250 mL
c) Solution: CaCl2 x 2 H2O 9.10 g; Deionised water ad 250 mL
d) Solution: FeCl3 x 6 H2O 0.25 g; Deionised water ad 1000 mL
- Ration of ingredients: 1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was about 8.7 mg/L at about 22 °C.
- Temperature (room): 22 ± 2 °C
- Temperature (flasks): 21.1 - 22.6 °C
- Kept in the dark: yes.
- Oxygen concentration: 8.7 mg/L, at the start of the test.
- pH: 7.28, at the start of the test.

TEST SYSTEM
- Preperation of test flasks: Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.
- Preperation of test solution: Before the preparation of the respective test solutions an aqueous stock solution of test material with a concentration of 30 mg/L was prepared and diluted accordingly. The test material concentration in the test solutions was 3.0 mg/L. The chosen test material concentration was based on the measured chemical oxygen demand (COD): 1.97 ±0.04 mg O2/mg test material and on the performed 14-d preliminary test.
- Test vessel: BOD bottles (300 ml) with special neck and glass stoppers.
- Exposure vessels: Microbial inoculum 0.5 mL/L, test material 3.0 mg/L.
- Number of culture flasks/concentration: 10 plus 4 flasks were prepared for each test system

SAMPLING AND MEASUREMENTS
- COD: determined in using a COD measuring system.
- Oxygen: The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 1, 7, 11, 14, 21 and 28.
- Temperature: Measured continuously and registered on weekdays.

CONTROL AND BLANK SYSTEM
- Procedural control: Microbial inoculum 0.5 mL/L, reference substance 3.6 mg/L.
- Inoculum control: Filtered microbial inoculum 0.5 mL/L.
- Toxicity control: Microbial inoculum 0.5 mL/L, test material 3.0 mg/L, reference substance 3.6 mg/L.

Reference substance:

benzoic acid, sodium salt

Parameter:

other: COD

Value:

64.3

Sampling time:

14 d

Parameter:

other: COD

Value:

63.5

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test material reached a mean of 63.5 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of COD and this level has to be reached in a 10-day window within the 28-day period of the test. However taking into account that the test material was sufficiently degraded to a mean of 64.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test item can be considered to be ready biodegradable.

Percentage biodegradability of the test material, reference substance and toxicity control can be seen in Table 'Percentage Biodegradation at Different Time Intervals during the Exposure Period' and the attachment in the 'Overall remarks, attachments' section.

Results with reference substance:

Sodium benzoate was sufficiently degraded to a mean of 77.5 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

Toxicity:

In the toxicity control containing both, the test material and the reference substance, a mean of 45.0 % biodegradation was noted within 14 days and 47.9 % biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 3.0 mg/L on the activated sludge microorganisms because degradation was > 25 % within 14 days.

 

Dissolved Oxygen Concentration at Different Time Intervals during the Exposure Period

Treatment	Concentration (mg/L)	Flask No.	mg O2/L after n days of exposure
			0	1	7	11	14	21	28
Test Material	3.0	1a	8.6	7.8	5.0	4.7	4.3	4.0	3.8
		1b	8.6	7.9	4.7	4.3	4.0	3.8	3.5
		mean	8.60	7.85	4.85	4.50	4.15	3.90	3.65
Reference Substance	3.6	2a	8.6	8.3	4.0	3.6	3.3	3.0	2.7
		2b	8.6	8.3	4.0	3.5	3.3	3.1	2.7
		mean	8.60	8.30	4.00	3.55	3.30	3.05	2.70
Inoculum Control	-	3a	8.7	8.5	8.2	7.9	8.0	7.9	7.3
		3b	8.6	8.5	8.0	7.8	8.0	7.6	7.6
		mean	8.65	8.50	8.10	7.85	8.00	7.75	7.45
Toxicity Control	Test material 3.0 Ref. Sub. 3.6	4a	8.6	8.1	3.5	3.0	2.5	1.9	1.5
		4b	8.6	8.1	3.8	3.0	2.6	2.1	1.8
		mean	8.60	8.10	3.65	3.00	2.55	2.00	1.65

 

Oxygen Depletion at Different Time Intervals during the Exposure Period

Treatment	Concentration (mg/L)	Flask No.	mg O2/L after n days of exposure
			1	7	11	14	21	28
Test Material	3.0	1a	0.65	3.05	3.10	3.65	3.70	3.60
		1b	0.55	3.35	3.50	3.95	3.90	3.90
Reference Substance	3.6	2a	0.15	4.05	4.20	4.65	4.70	4.70
		2b	0.15	4.05	4.30	4.65	4.60	4.70
Toxicity Control	Test material 3.0 Ref. Sub. 3.6	4a	0.35	4.55	4.80	5.45	5.80	5.90
		4b	0.35	4.25	4.80	5.35	5.60	5.60

 

Total Oxidised Nitrogen at concentrations at Different Time Intervals during the Exposure Period

Treatment	Concentration (mg/L)	Flask No.	mg total oxidised N/L after n days of exposure
			0	1	7	11	14	21	28
Test Material	3.0	1a	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
		1b	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Inoculum Control	-	3a	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
		3b	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Toxicity Control	Test material 3.0 Ref. Sub. 3.6	4a	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
		4b	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ

LOQ: 4.43 mg NO₃/mL; LOQ: 0.03 mg NO₂/mL; 

 

BOD at Different Time Intervals during the Exposure Period

Treatment	Concentration (mg/L)	Flask No.	BOD after n days of exposure
			1	7	11	14	21	28
Test Material	3.0	1a	0.22	1.02	1.03	1.22	1.23	1.20
		1b	0.18	1.12	1.17	1.32	1.30	1.30
Reference Substance	3.6	2a	0.04	1.13	1.17	1.29	1.31	1.31
		2b	0.04	1.13	1.19	1.29	1.28	1.31
Toxicity Control	Test material 3.0 Ref. Sub. 3.6	4a	0.05	0.69	0.73	0.83	0.88	0.89
		4b	0.05	0.64	0.73	0.81	0.85	0.85

 

Percentage Biodegradation at Different Time Intervals during the Exposure Period

Treatment	Concentration (mg/L)	Flask No.	BOD after n days of exposure
			1	7	11	14	21	28
Test Material	3.0	1a	11.0	51.6	52.5	61.8	62.6	60.9
		1b	9.3	56.7	59.2	66.8	66.0	66.0
		mean	10.2	54.1	55.8	64.3	64.3	63.5
Reference Substance	3.6	2a	2.5	67.5	70.0	77.5	78.3	78.3
		2b	2.5	67.5	71.7	77.5	76.7	78.3
		mean	2.5	67.5	70.8	77.5	77.5	78.3
Toxicity Control	Test material 3.0 Ref. Sub. 3.6	4a	2.9	37.9	40.0	45.4	48.3	49.2
		4b	2.9	35.4	40.0	44.6	46.7	46.7
		mean	2.9	36.7	40.0	45.0	47.5	47.9

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the test, the test material was determined to have a percentage biodegradation of 63.5 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of COD and this level has to be reached in a 10-day window within the 28-day period of the test. However taking into account that the test material was sufficiently degraded to a mean of 64.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.
In the toxicity control containing both, the test material and the reference substance Sodium benzoate, a mean of 45.0 % biodegradation was noted within 14 days and 47.9 % biodegradation after 28 days of incubation. According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 3.0 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Executive summary:

The ready biodegradability of the test material was determined in a Closed Bottle Test over 28 days using sewage obtained from a domestic sewage plant. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 301 D, EPA OPTS 835.3110, and EU Method C.4-E.

Under the conditions of the test, the test material was determined to have a percentage biodegradation of 63.5 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of COD and this level has to be reached in a 10-day window within the 28-day period of the test. However taking into account that the test material was sufficiently degraded to a mean of 64.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.

In the toxicity control containing both, the test material and the reference substance Sodium benzoate, a mean of 45.0 % biodegradation was noted within 14 days and 47.9 % biodegradation after 28 days of incubation. According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 3.0 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Description of key information

Ready biodegradable, 63.5 % biodegradation after 28 days, OECD 301 D, EPA OPTS 835.3110, and EU Method C.4-E, Sipos (2013a).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In the key study (Sipos, 2013a) the ready biodegradability of the test material was determined in a Closed Bottle Test over 28 days using sewage obtained from a domestic sewage plant. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 301 D, EPA OPTS 835.3110, and EU Method C.4-E.

Under the conditions of the test, the test material was determined to have a percentage biodegradation of 63.5 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of COD and this level has to be reached in a 10-day window within the 28-day period of the test. However taking into account that the test material was sufficiently degraded to a mean of 64.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.

In the toxicity control containing both, the test material and the reference substance Sodium benzoate, a mean of 45.0 % biodegradation was noted within 14 days and 47.9 % biodegradation after 28 days of incubation. According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 3.0 mg/L on the activated sludge microorganisms because degradation was > 25 % within 14 days.