Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

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:

From 05 September 2019 to 03 October 2019

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Species/Origin:
Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.

Conditioning:
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.

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 102.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

(A) Test Units
Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System
Principle: The test flasks prepared according paragraph 6.6 were incubated at 22°C * 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System. The test flasks were closed gas-tight by a measuring head. 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.
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.

(B) Test Conditions
Surrounding Type: Climatised chamber
Temperature: 22°C ± 1°C
Light Conditions: Darkness
pH-Value of Test Solutions: 7.6 (measured at the start of the test), 7.1 to 7.6 (measured at the end of the test)
Recording: Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and were not reported.)

(C) Test Water
Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
The pH was adjusted to pH 7.5 with 1M HCl solution.

(D) Course of the Test
Preparation of Test Flasks: The amounts of test substance and reference substance 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 substance.
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.
Test Duration: 28 days

(E) Test Parameters
Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System).
Temperature: The temperature was recorded by means of the automated software AMR Wincontrol©.
pH-Value: pH-values were measured in procedure control, a separately prepared test flask with test substance (to prevent loss of test substance in the test flasks) and a separately prepared test flask without test substance (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

(F) Result Evaluation
Definitions:
ThODNH4:Theoretical Oxygen Demand
ThODNH4 is the total amount of oxygen required to oxidise a chemical completely. It is calculated from the molecular formula (assuming no nitrification occurs) and expressed as mg oxygen required per mg test substance.

ThODNO3:Theoretical Oxygen Demand with Nitrification
ThODNO3 is the total amount of oxygen required to oxidise a chemical completely, assuming nitrification occurs and expressed as mg oxygen required per mg test substance.

BOD: Biochemical Oxygen Demand
BOD is the amount of oxygen consumed by microorganisms when metabolising a test substance; also expressed as mg oxygen uptake per mg test substance/L.
Conversion Factor: Conversion Factor is necessary for the calculation of the BOD and depends on the final volume in the test flasks and is predetermined by the BSB/BOD-Sensor-System supplier. At a test volume of 244 ml the conversion factor is 5.
10-day window: The 10 day window is the 10 day period immediately following the attainment of 10 % biodegradation
BOD [mg/L] = Measured data * conversion factor
BODControl adjusted = BOD of test substance (or reference substance) – mean BOD of inoculum controls
ThOD [mg O2/L] = Test substance or reference substance [mg] * ThOD [mg O2/mg test substance or reference substance] / 0.244L

The percentage biodegradation of the test substance and of the reference substance sodium benzoate is calculated as:
% degradation = BODControl adjusted [mg/L] * 100 / ThOD [mg O2/L]
For the toxicity control, the ThOD is the sum of the ThOD of the test substance and the ThOD of the reference substance weighed in the toxicity control flask.

(G) Validity Criteria of the Study
Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 2.5 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 substance flasks at the end of the test was 7.3 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

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

Test Substance: The difference of duplicate values for the degradation of the test substance at the plateau, 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 day 28 was 4%. The validity criterion was fulfilled.

Toxicity Control: If in a toxicity test, containing both the test substance and a reference substance less than 25% biodegradation (based on ThODNH4) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 35% at day 14; the test substance was not inhibitory.

Reference substance:

benzoic acid, sodium salt

Remarks:

Batch No.:SLBW2610, Retest date: November 2021

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

test substance

Value:

ca. 37

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

sodium benzoate

Value:

ca. 78

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

toxicity control

Value:

ca. 42

Sampling time:

28 d

Details on results:

Results and Discussion
(a) Biodegradation of Test Substance
Percentage Biodegradation: The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test substance of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test substance reaches at least 10% degradation. The 10-day window began on day 3 after application; the mean value was calculated to be 12% biodegradation (ThODNH4, ThODNO3). Therefore, the end of the 10-day window was day 13. After correction for the mean biochemical oxygen demand of the inoculum controls the mean biodegradation percentage based on ThODNO3 and ThODNH4 at the end of the 10-day window was 26%; the criterion of the 10 day window was not passed. The mean biodegradation percentage at the end of the 28-day exposure period was 37% (ThODNO3, ThODNH4).
Conclusion: The degradation rate of Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation never reached 60%. Therefore, Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation is considered to be not readily biodegradable.

(b) Biodegradation of Reference Substance Sodium Benzoate
Percentage Biodegradation: The reference substance sodium benzoate was sufficiently degraded to 75% after 14 days and to 78% after 28 days of incubation.
Conclusion: The percentage biodegradation of the reference substance confirms the suitability of the used aerobic activated sludge inoculum.

(c) Biodegradation in the Toxicity Control
Percentage Biodegradation: In the toxicity control containing both, the test substance and the reference substance sodium benzoate, 35% (ThODNH4) biodegradation was noted within 14 days and 42% (ThODNH4) biodegradation after 28 days of incubation (35% and 42% based on ThODNO3).
Conclusion: According to the test guidelines the test substance can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

(d) Abiotic Control
Oxygen Demand: The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test substance and toxicity control.

Results with reference substance:

The reference substance sodium benzoate was sufficiently degraded to 75% after 14 days and to 78% after 28 days of incubation.

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

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	15	5	0	0	30	0	50
2	25	25	0	0	70	0	95
3	40	40	0	5	95	0	115
4	45	50	0	5	105	0	120
5	50	60	0	5	110	0	135
6	55	65	0	5	115	0	140
7	60	70	0	5	120	0	145
8	60	75	0	5	120	0	155
9	65	80	0	5	125	0	160
10	65	85	0	5	125	0	165
11	70	90	0	5	125	0	170
12	75	90	0	5	125	0	170
13	75	95	0	5	130	0	170
14	75	100	0	5	130	0	175
15	80	100	0	5	130	0	175
16	80	105	0	5	130	0	180
17	80	110	0	5	135	0	180
18	85	115	0	5	135	0	185
19	90	115	0	5	135	0	185
20	95	120	0	5	135	0	190
21	95	125	0	5	135	0	195
22	100	125	0	5	135	0	195
23	105	125	0	5	135	0	195
24	105	130	0	5	135	0	200
25	110	130	0	5	135	0	200
26	110	130	0	5	135	0	205
27	115	130	0	5	135	0	205
28	115	130	0	5	135	0	205

Flasks 1 and 2: Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation; 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 Substance, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time	Percentage Biodegradation
(Days)	Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	5	2	18	10
2	8	8	41	19
3	12	12	54	23
4	13	15	60	24
5	15	18	63	27
6	16	19	66	28
7	18	21	69	29
8	18	22	69	31
9	19	24	72	32
10	19	25	72	33
11	21	27	72	34
12	22	27	72	34
13	22	29	75	34
14	22	30	75	35
15	24	30	75	35
16	24	32	75	36
17	24	33	78	36
18	25	35	78	37
19	27	35	78	37
20	29	36	78	38
21	29	38	78	39
22	30	38	78	39
23	32	38	78	39
24	32	39	78	41
25	33	39	78	41
26	33	39	78	42
27	35	39	78	42
28	35	39	78	42

¹ThOD_NH4of Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation: 3.149 mg O₂/mg test substance
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference substance

Table 3: Percentage Biodegradation of Test Substance and of the Toxicity Control based on ThOD_NO3and of Sodium Benzoate based on ThOD_NH4

Time	Percentage Biodegradation
(Days)	Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	5	2	18	10
2	8	8	41	19
3	12	12	54	23
4	13	15	60	24
5	15	18	63	27
6	16	19	66	28
7	18	21	69	29
8	18	22	69	31
9	19	24	72	32
10	19	25	72	33
11	21	27	72	34
12	22	27	72	34
13	22	29	75	34
14	22	30	75	35
15	24	30	75	35
16	24	32	75	36
17	24	33	78	36
18	25	35	78	37
19	27	35	78	37
20	29	36	78	38
21	29	38	78	39
22	30	38	78	39
23	32	38	78	39
24	32	39	78	40
25	33	39	78	40
26	33	39	78	42
27	35	39	78	42
28	35	39	78	42

¹ThOD_NO3of Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and distillation: 3.152 mg O₂/mg test substance
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference substance

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the test substance is not readily biodegradable, but based on the percentage degradation, which exceeds 20%, it can be considered to undergo inherent primary biodegradability.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ using the manometric respirometry method according to OECD Guideline 301F, in compliance with GLP. Microorganisms from a domestic wastewater treatment plant (aerobic activated sludge) was used for the study along with test substance loading rate of 102.9 mg/L corresponding to an oxygen demand of about 323.9 mg/L (ThODNH4, Theoretical Oxygen Demand) and 324.2 mg/L (ThODN03, Theoretical Oxygen Demand with Nitrification). Sodium benzoate was used as reference substance with loading rate of 102.5 mg/L corresponding to an oxygen demand of about 170.7 mg/L (ThODNH4). The test substance 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 substance sodium benzoate was tested simultaneously under the same conditions as the test substance and functioned as a procedure control. Degradation rate of test substance calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The mean biodegradation of 10% of test substance was reached at day 3 (ThODNH4, ThODNO3). At the end of the 10-day window at day 13, the mean degradation of test substance was 26% (ThODNH4, ThODNO3) and therefore the 10-day window criterion was not passed. The mean biodegradation at test end after 28 days was 37% (ThODNH4, ThODNO3). Therefore, test substance is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. The reference substance sodium benzoate was sufficiently degraded to 75% after 14 days and to 78% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test substance and the reference substance sodium benzoate, 35% (ThODNH4, ThODNO3) biodegradation was noted within 14 days and 42% (ThODNH4, ThODNO3) biodegradation after 28 days of incubation. According to the test guidelines, the test substance can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days (Hammesfahr, 2019). Under the study conditions, the test substance was not readily biodegradable, but based on the percentage degradation which exceeds 20%, it can be considered to undergo inherent primary biodegradability (OECD, 2003). Further, given the very low water solubility and intermediate use of the test substance, no direct and indirect exposure of the test substance to the environment is expected, hence no concern around potential persistence in water is expected.  

Description of key information

The test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is not readily biodegradable, but based on its percentage degradation, which exceeds 20%, it can be considered to undergo inherent primary biodegradation. Further, given the very low water solubility and intermediate use of the test substance, no direct or indirect exposure of the test substance to the environment is expected, indicating lack of persistence potential.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ using the manometric respirometry method according to OECD Guideline 301F, in compliance with GLP. Microorganisms from a domestic wastewater treatment plant (aerobic activated sludge) was used for the study along with test substance loading rate of 102.9 mg/L corresponding to an oxygen demand of about 323.9 mg/L (ThODNH4, Theoretical Oxygen Demand) and 324.2 mg/L (ThODN03, Theoretical Oxygen Demand with Nitrification). Sodium benzoate was used as reference substance with loading rate of 102.5 mg/L corresponding to an oxygen demand of about 170.7 mg/L (ThODNH4). The test substance 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 substance sodium benzoate was tested simultaneously under the same conditions as the test substance and functioned as a procedure control. Degradation rate of test substance calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The mean biodegradation of 10% of test substance was reached at day 3 (ThODNH4, ThODNO3). At the end of the 10-day window at day 13, the mean degradation of test substance was 26% (ThODNH4, ThODNO3) and therefore the 10-day window criterion was not passed. The mean biodegradation at test end after 28 days was 37% (ThODNH4, ThODNO3). Therefore, test substance is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. The reference substance sodium benzoate was sufficiently degraded to 75% after 14 days and to 78% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test substance and the reference substance sodium benzoate, 35% (ThODNH4, ThODNO3) biodegradation was noted within 14 days and 42% (ThODNH4, ThODNO3) biodegradation after 28 days of incubation. According to the test guidelines, the test substance can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days (Hammesfahr, 2019). Under the study conditions, the test substance was not readily biodegradable, but based on the percentage degradation which exceeds 20%, it can be considered to undergo inherent primary biodegradability (OECD, 2003). Further, given the very low water solubility and intermediate use of the test substance, no direct and indirect exposure of the test substance to the environment is expected, indicating lack of persistence potential.