2-hydroxy-N-((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)octadecanamide

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:

2017-08-31 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1992-07-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ceramide IIIB

IUPAC Name:

Ceramide IIIB

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

The test item was weighed out for each replicate and directly transferred into the test vessels with 250 mL of the inoculated test medium, using a 250 mL measuring flask.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, 31137 Hildesheim, Germany
- Receipt: 2017-08-24
- Pretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was re-suspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 6 days. 10 mL/L were used to initiate inoculation.
- Colony forming units in the test vessels: approximately 1.00E+07 – 1.00E+08 CFU/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

18 mg/L

Based on:

test mat.

Remarks:

ThOD: 2.80 mg O2/mg; ThOD NO3: 2.91 mg/ O2/mg

Initial test substance concentration 2

Initial conc.:

50.4 g/L

Based on:

ThOD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 F
- Test temperature: 20.0 - 20.3 °C
- pH: start: 7.73, end: 7.67 - 7.93
- pH adjusted: no
- Continuous darkness: yes
TEST SYSTEM
- Culturing apparatus: dark brown bottles, 250ml
- Number of culture flasks/concentration:
two for the inoculum control (C1, C2)
one for the functional control (R1)
two for the test item (P1, P2)
one for the toxicity control (T1)
- Method used to create aerobic conditions: aeration 6 days
- Measuring equipment:
OxiTop OC110 controller with software Achat OC, WTW
OxiTop measuring heads, WTW
Climatic exposure test cabinet, RUMED Rubarth Apparate GmbH
pH-Meter, Multi 350i, WTW
Datalogger 174 T, Testo
Analytical balance, Sartorius
Balance, Kern
Multipette X-Stream, eppendorf
Spectralphotometer NANOCOLOR® UV/VIS, Macherey-Nagel
Test Kits: Nitrat 50, Nitrit 4, Nitrit 2, all by Macherey-Nagel
Various pipettes
CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum Control: Test medium without test and / or reference item, 2 vessels
- Abiotic sterile control: functional control one vessel
- Toxicity control: one vessel
For further details on test system, please see section “any other information on materials and methods incl. tables”.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

45 mg/L

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

62

Sampling time:

28 d

Remarks on result:

other: biodegradation rate as % BOD

Details on results:

For a detailed description of results, please see section "any other information on results incl. tables".

BOD5 / COD results

Results with reference substance:

The percentage degradation of the reference substance sodium benzoate reached the pass level of 60 % within 3 days.

Any other information on results incl. tables

Results

Theoretical Oxygen Demand

The biodegradation in % was calculated based on the ThOD_NO3 of 2.91 mg O₂/mg test item.

Colony Forming Units of the Inoculum

Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.67E+09 CFU/L. The CFU concentration of the inoculum corresponds to approx. 1.67E+07 CFU/L in the final test solution.

Results of the Functional Control

The pass level for ready biodegradation (≥ 60 % degradation) was reached within 3 days. The validity criterion that the degradation should be ≥ 60 % after 14 d was fulfilled. Stages of Biodegradation: The adaptation phase changed to degradation phase within 2 days (degradation > 10 %) and the biodegradation came to a maximum of 100 % on day 19.

Results of the Toxicity Control

In the toxicity control the biodegradation achieved 69 % after 14 days. After 28 days the biodegradation came to 83 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Course and Stages of Biodegradation of Functional Control and Test Item

The biodegradation of the test item is shown in Table 1 and graphically presented in Figure 1 in comparison to the readily degradable functional control and the toxicity control. The mean of replicates reached the 10 % level (beginning of biodegradation) on day 5 and the 60 % pass level on day 27. The pass level of the 10-day-window was not fulfilled (< 40 % on Day 15). The mean biodegradation on day 28 was 62 %. For the calculation of the biodegradation, the ThOD_NO3 of 52.4 mg O₂/L (i.e. with nitrification) was considered, since the concentration of nitrate increased during the test.

After a test period of 28 days, the test item is classified as readily biodegradable within the 28 day period of the study.

Table1: Biodegradation [%] of the Test Itemin Comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 18mg/L	1	18	36	51	64
	2	17	34	46	60
Functional Control 45 mg/L		85	96	100	100
Toxicity Control 18mg/L Test Item + 45 mg/L Reference Item		56	69	77	83

 

Table2:     Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control and Toxicity Control

		BOD [mg O2/L]
Date	[d]	Inoculum Control	Functional Control		Test Item				Toxicity Control Test Item + Reference Item
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
2017-09-01	1	2.5	8.7	6.2	4.2	1.7	3.3	0.9	11.8	9.3
2017-09-02	2	3.0	45.8	42.9	5.3	2.3	4.4	1.5	48.8	45.8
2017-09-03	3	3.1	56.6	53.5	6.5	3.4	5.4	2.3	59.3	56.2
2017-09-04	4	3.0	61.4	58.4	8.1	5.1	7.6	4.6	66.0	62.9
2017-09-05	5	3.3	64.4	61.0	9.8	6.5	9.1	5.8	68.8	65.5
2017-09-06	6	3.2	65.7	62.5	11.3	8.1	10.5	7.4	71.4	68.3
2017-09-07	7	3.3	67.5	64.2	12.9	9.6	12.1	8.7	74.7	71.4
2017-09-08	8	4.3	70.6	66.3	15.1	10.8	14.6	10.3	78.8	74.5
2017-09-09	9	4.5	71.3	66.9	15.6	11.1	15.6	11.1	80.8	76.4
2017-09-10	10	3.6	71.0	67.4	16.1	12.5	16.0	12.4	82.6	79.0
2017-09-11	11	4.6	73.3	68.8	18.7	14.1	18.7	14.1	86.3	81.8
2017-09-12	12	4.5	74.6	70.1	20.3	15.8	20.5	15.9	89.3	84.8
2017-09-13	13	4.6	75.1	70.5	22.3	17.7	21.2	16.6	90.6	86.0
2017-09-14	14	5.3	77.2	71.9	24.2	18.9	23.1	17.8	93.6	88.4
2017-09-15	15	4.7	77.5	72.8	25.2	20.4	24.0	19.2	96.0	91.3
2017-09-16	16	4.9	77.9	73.0	26.0	21.1	24.4	19.5	95.9	91.0
2017-09-17	17	6.1	80.8	74.8	29.2	23.1	27.7	21.6	100.3	94.2
2017-09-18	18	5.5	80.1	74.5	29.5	23.9	27.3	21.7	99.6	94.1
2017-09-19	19	6.1	81.4	75.2	31.0	24.9	29.2	23.0	102.7	96.6
2017-09-20	20	6.0	81.9	75.9	32.1	26.1	29.7	23.7	103.7	97.7
2017-09-21	21	5.8	81.7	75.9	32.4	26.6	30.0	24.2	104.1	98.3
2017-09-22	22	6.1	82.6	76.4	34.0	27.9	32.0	25.8	106.2	100.1
2017-09-23	23	9.1	84.8	75.7	37.2	28.1	34.8	25.7	109.2	100.1
2017-09-24	24	6.4	83.2	76.8	36.1	29.6	33.8	27.4	108.7	102.3
2017-09-25	25	6.6	84.2	77.6	37.5	30.9	35.2	28.6	110.0	103.4
2017-09-26	26	7.1	84.8	77.6	38.9	31.8	36.5	29.4	111.6	104.5
2017-09-27	27	6.9	84.9	78.0	39.4	32.5	37.6	30.7	112.6	105.8
2017-09-28	28	7.5	85.4	78.0	41.1	33.6	39.1	31.7	113.8	106.3

 

Table3:     Biodegradation [%] of the Test Itemin Comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
Date	[d]	Functional Control	Test Item		Toxicity Control Test Item + Reference Item
		R1	P1	P2	T1
2017-09-01	1	8	3	2	7
2017-09-02	2	57	4	3	36
2017-09-03	3	71	6	4	44
2017-09-04	4	78	10	9	49
2017-09-05	5	81	12	11	51
2017-09-06	6	83	16	14	53
2017-09-07	7	85	18	17	56
2017-09-08	8	88	21	20	58
2017-09-09	9	89	21	21	60
2017-09-10	10	90	24	24	62
2017-09-11	11	91	27	27	64
2017-09-12	12	93	30	30	66
2017-09-13	13	94	34	32	67
2017-09-14	14	96	36	34	69
2017-09-15	15	97	39	37	72
2017-09-16	16	97	40	37	71
2017-09-17	17	99	44	41	74
2017-09-18	18	99	46	41	74
2017-09-19	19	100	47	44	76
2017-09-20	20	100	50	45	77
2017-09-21	21	100	51	46	77
2017-09-22	22	100	53	49	78
2017-09-23	23	100	54	49	78
2017-09-24	24	100	57	52	80
2017-09-25	25	100	59	55	81
2017-09-26	26	100	61	56	82
2017-09-27	27	100	62	59	83
2017-09-28	28	100	64	60	83

 

pH-Values at Test Start and Test End

The pH-values of the samples were measured at test start and test end (day 28). The results are given inTable 4.

 

Table4:     pH-Values at Test Start and Test End. 1), 2) = First, second replicate

	pH-Value
	Start	End
Inoculum Control	7.73	1) 7.67
		2) 7.66
Functional Control	7.74	7.83
Test Item	7.75	1) 7.69
		2) 7.65
Toxicity Control	7.74	7.93

 

Determination of oxidized Nitrogen Species at Test End

At test end, the concentration of nitrite and nitrate was determined by photometric measurement from aliquots of the control and test item replicates (Table 5). The corresponding oxygen consumption was calculated with the factors 3.43 for nitrite and 4.57 for nitrate, as given in
OECD 301F. The increase of the nitrate concentration indicates nitrification. Therefore, the values of the biodegradation were calculated based upon the ThOD_NO3.

 

Table5:     Nitrite and Nitrate Concentrations at Test End

	Nitrite- N	Nitrate-N	corresponding Oxygen consumption	Biodegradation calculated without nitrification	Biodegradation calculated with nitrification
	[mg/L]	[mg/L]	[mg/L]	[%]	[%]
Inoculum Control C1	0	0	0	-	-
Inoculum Control C2	0.003	0	0	-	-
Test item repl. P1	0	0.9	4.1	67	64
Test item repl. P2	0	0.8	3.7	63	60

 

Validity Criteria

 

The study was performed according to OECD Guideline 301 F and GLP principles. The validity criteria were fulfilled according to the guideline:

 

·       The percentage degradation of the functional control reached the pass level of 60 %
within 3 days (validity criterion: 60 % after 14 days).

·       The mean oxygen depletion in the inoculum control was 7.5 mg oxygen/L on day 28
(validity criterion: < 60 mg oxygen/L).

·       The pH-value of all test replicates was in the range of 6.0 - 8.5 at the end of the test.

·       The difference of extremes of replicate values of removal of the test item at the end of the test was less than 20 % (4% difference on day 28).

·       The percentage degradation of the toxicity control reached the pass level of 25 % within
2 days (validity criterion: 25 % after 14 days).

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

After a test period of 28 days, the test item was classified as readily biodegradable within the 28 day period of the study but failed the 10-day window.

Executive summary:

The ready biodegradability of Ceramide IIIB was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F. The concentration of Ceramide IIIB selected as appropriate was 18 mg/L, corresponding to a ThOD of 50.4 mg O₂/L (52.4 mg O₂/L with nitrification) per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 7.5 mg O₂/L on day 28.

Sodium benzoate was used as functional control. The pass level > 60 % was reached within 3 days. The biodegradation reached a maximum of 100 % degradation on day 19.

In the toxicity control containing both test and reference item 69 % degradation occurred within 14 days. After 28 days the biodegradation came to 83 %. The degradation of the reference item was not inhibited by Ceramide IIIB.

The mean of replicates reached the 10 % level (beginning of biodegradation) on day 5 and the 60 % pass level on day 27. The mean biodegradation on day 28 was 62 %. For the calculation of the biodegradation, the ThOD_NO3 of 52.4 mg O₂/L (i.e. with nitrification) was considered, since the concentration of nitrate increased during the test.

The validity criteria of the guideline are fulfilled.

Ceramide IIIB is classified as readily biodegradable within the 28 day period of the study, but failing the 10-day window.