1-O-β-(2 ,3-di-O-alka(e)noyl-6 -O-acetyl-D-mannopyranosyl)-D-erythritol

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:

14 November 2018 - 12 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17 July 1992

Deviations:

yes

Remarks:

HPLC was used to quantitatively determine residual test item and any degradation products

Qualifier:

according to guideline

Guideline:

other: II: Method for Testing Biodegradability of Chemical Substances by Microorganisms (301F) stipulated in the "Testing Methods for New Chemical Substances"

Version / remarks:

No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5 , Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; latest revision, March 29, 2018, No. 0329-13, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; No. 20180326-1, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 1803293, Environmental Policy Bureau, Ministry of the Environment, Japan

Deviations:

not specified

Principles of method if other than guideline:

Utilisation of HPLC to quantitatively determine residual test item and any degradation products is specific to the Japanese biodegradation test method referenced in ‘Materials and methods – Test guidelines’. Use of HPLC does not feature in the OECD 301 F guideline.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Sewage treatment plant treating predomininantly domestic sewage in Kurume-shi, Fukuoka, Japan. Sample date: 14 November 2018
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Aerated
- Storage length: < 24 h
- Preparation of inoculum for exposure: The activated sludge was obtained from an aeration tank of the Kurume-shi sewage treatment plant and aerated at approx. 22 ºC until use in the test. 3.38 mL of activated sludge was added to the test vessel on the basis of the concentration of suspended solid in the activated sludge, determined in accordance with the Japanese Industrial Standards Method K 0102:2016, Section 14.1. The determined concentration was 2660 mg/L. The mineral medium (5 L) was prepared as the follows: 10 mL of solution (a) and 1 mL each of solutions (b), (c) and (d) as prescribed in the OECD 301 test guideline (Section 5) were made up to 1 L with purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical), then the pH adjusted to 7.4. To prepare the test solutions, 30 mg of test item (accurately weighed with an electronic analytical balance) and 5 mL of mineral was added to individual glass test tubes. It was then heated at approx. 60 ºC for 5 minutes in a water bath and strongly stirred for 1 minute with a vortex mixer. This was repeated once more. The suspension in each glass test tube was added to a test vessel along with the mineral medium (subtracting the volume 3.38 mL of activated sludge from 245 mL) while washing with 50 mL of the mineral medium, so that the concentration of the test item reached 100 mg/L.
- Pretreatment: N/A
- Concentration of sludge: 30 mg suspended solids/L
- Initial cell/biomass concentration: N/A
- Water filtered: yes/no: No
- Type and size of filter used, if any: N/A

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: As per the OECD 301 guideline, prepared using purified water
- Additional substrate: No
- Solubilising agent (type and concentration if used): No
- Test temperature: 22 ± 1 ºC
- pH: pH measurements of test solutions were not taken at preparation, because the test item was not dissolved at 100 mg/L in water.
- pH adjusted: Yes
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes
- Other: N/A

TEST SYSTEM
- Culturing apparatus: Glass vessels
- Number of culture flasks/concentration: Six vessels (2 for the test solution, 2 for inoculum blanks, 1 for procedure control, 1 for toxicity control)
- Method used to create aerobic conditions: Not reported
- Method used to create anaerobic conditions: N/A
- Measuring equipment: Closed system oxygen consumption measuring apparatus (No. CM-062). Total organic carbon (TOC) analyzer (No. TOC-013): TC-IC measurement method; temperature of furnace: 680 ºC; flow rate: 150 mL/min; injection volume 50 µL; LOQ: 1.0 mgC/L
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Soda lime No.1 for absorption of CO2, FUJIFILM Wako Pure Chemical
- Other: Each test solution was stirred using a stirrer

SAMPLING
- Sampling frequency: During the incubation period, the BOD of the test solutions was measured continuously.
- Sampling method: Closed system oxygen consumption measuring apparatus
- Sterility check if applicable: N/A
- Sample storage before analysis: Not reported
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test solution (control blank) (n=2: test vessels 3 and 4). In each test vessel, the mineral medium (subtracting the volume 3.38 mL of activated sludge from 300 mL) was added.
- Abiotic sterile control: Not performed
- Toxicity control: Test solution (sluge + test item + sodium benzoate) (n=1, test vessel 6). In one test vessel, 30 mg of sodium benzoate (accurately weighed using an analytical electronic balance) was added to the mineral medium (by subtracting the volume 3.38 mL of activated sludge from 245 mL). The suspension of the test item was then prepared similarly to that described previously in 'Details on inoculum' and was added whilst washing with 50 mL of the mineral medium so that the concentration of the test item and sodium benzoate reached 100 mg/L.
- Other: Procedure control: Test solution (sludge + sodium benzoate)(n=1: test vessel 5). In one test vessel, 30 mg of sodium benzoate (accurately weighed using an analytical electronic balance) was added to the mineral medium (by subtracting the volume 3.38 mL of activated sludge from 300 mL) so that the concentration of sodium benzoate reached 100 mg/L.

STATISTICAL METHODS:
The percentage by DOC was not calculated because the test item was not dissolved at 10 mg/L in water.

Calculation for percentage biodegradation by BOD:
Degradation = (BOD - B average) / (ThOD) x 100

where B average = Average of BOD control blank

ThOD of test item = 59.4 mg
C26H44O10 + 32.00 O2 -> 26.00 CO2 + 22.00 H2O
ThOD = additive amount of test item x 32.00 O2 / C26H44O10 = 59.4 mg

TOD of sodium benzoate: 50.1 mg
C7H5NaO2 + 7.50 O2 -> 7.00 CO2 +2.50 H2O + 0.5 Na2O
ThOD = additive amount of sodium benzoate x 7.50 O2 / C7H5NaO2 = 50.1

ThOD of test item + sodium benzoate = 59.4 + 50.1 = 109.5 mg

Calculation of percentage biodegradation of test item (the residual amount was set as 0 and the calculation carried out because the analytical value obtained was < LOD during quantititive analysis:
% biodegradation = [(Amount of test item added - R) / Amount of test item added] x 100

where R = Residual amount of test item (mg)
R = I x [A(sludge + test item)- Average of A(Control blank)) / A(Standard)] x (B/C) / (D/100) / 1000

where
I = Concentration of standard solution (300 mg/L)
A = peak area (µV sec)
B = final volume (100 mL)
C = ratio of portion used for analysis (270/300)
D = recovery rate (99.9 %)

Reference substance:

other: sodium benzoate

Remarks:

supplier: Nacalai Tesque

Preliminary study:

N/A

Test performance:

All validity criteria were met. The difference between extremes of replicate vaues of % biodegradation in test solutions on day 28 was 3 % (BOD), i.e. < 20 % as stipulated in the guideline. The % biodegradation by BOD in the toxicity control on day 14 (sludge + test item + reference substance) was 81 %, i.e. >= 25 % as stipulated in the guideline. The BOD in test solutions (control blank) on day 28 was 27 and 23 mg/L*

*Measurement values (8.2 and 6.8 mg) / [test volume (300 mL / 1000) = 27 and 23 mg/L

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

91

Sampling time:

28 d

Details on results:

Growth of the sludge was observed at the end of the incubation and no peaks corresponding to degradation products were detected on the HPLC chromatograms. DOC was barely detected. With an average 28 d biodegradation value of 91 % (BOD), the test item can be considered to be readily biodegradable under the conditions of this test. It was confirmed by HPLC analysis that 0 % test item remained and no degradation products were observed at the end of the test.

Results with reference substance:

In the solution containing sodium benzoate + sludge (procedural control), the percentage biodegradation by BOD was 97 % on day 14. As per the OECD 301F Guideline, the test system can be assumed to be functioning correctly if the percentage biodegradation is =>60 %.

Table 1. Visual observations

	Test solution	Appearance (visual)	pH
Start of incubation	Sludge + test item	Test item not dissolved. Test solution colourless.	-
	Control blank	No insoluble compound except the sludge was observed. Test solution colourless.	-
End of incubation	Sludge + test item	No insoluble compound except the sludge was observed. Growth of sludge was observed. Test solution colourless.	Vessel no. 1: 7.3 Vessel no. 2: 7.3
	Control blank	No insoluble compound except the sludge was observed. Test solution colourless.	Vessel no. 3: 7.3 Vessel no. 4: 7.3

Table 2. Analytical results of the test solutions on day 28

		Sludge + test item		Theoretical quantity^
		Vessel no. 1	Vessel no. 2
BOD	mg	52.7	54.6	59.4*
Detected amount and % detection of DOC	mgC	0.3	0.6	18.1**
	%	2	3	-
Residual amount and % residue of test item (HPLC)	mg	0	0	30.0
	%	0	0	-

_{*The average value of the test solutions (control blank) was subtracted from the values of the test solutions (sludge + test item)}

**Calculated from compositional formula C₂₆H₄₄O₁₀

Table 3. Percentage biodegration on day 28

	Sludge + test item
	Vessel no. 1	Vessel no. 2	Mean
% biodegradation (BOD)	89	92	91
% biodegradation (DOC)	The % biodegradation by DOC was not calculated because the test item cannot be dissolved in water at the test concentration (100 mg/L) or more
% biodegradation (HPLC)	100	100	100

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean percentage biodegradation on day 28 was 91 % by BOD. Growth of the sludge was observed at the end of the incubation and no peaks corresponding to degradation products were detected on the HPLC chromatograms, which supports the fact the parent was fully mineralised. DOC was barely detected as the test substance was insoluble. With an average 28 d biodegradation value of 91 % (BOD) the test item can be considered to be readily biodegradable under the conditions of this test.

Executive summary:

A ready biodegradability test was performed in accordance with OECD Test Guideline 301F (Manometric Respirometry; 1992) and the Japanese Guideline II: Method for Testing Biodegradability of Chemical Substances by Microorganisms (301F) stipulated in the "Testing Methods for New Chemical Substances".

The test item (concentration 100 mg/L) was added to two test vessels containing mineral medium inoculated with activated sludge (30 mg/L concentration of suspended solids) from a sewage treatment plant. Two inoculum blanks, a procedure blank and a toxicity control were run concurrently. Sodium benzoate was used as the reference substance in the toxicity control.

A closed system oxygen consumption measurer was used to monitor BOD of the test solutions continuously during the incubation period of 28 days.

at the end of the incubation period, the following analyses were conducted for the test solutions and control blank:

- Determination of DOC (using a TOC analyser)

- Determination of the test item and degradation products by HPLC (as described in the Japanese Guideline)

- Measurement of pH

The mean percentage biodegradation on day 28 was 91 % by BOD. Growth of the sludge was observed at the end of the incubation and no peaks corresponding to degradation products were detected on the HPLC chromatograms, which supports the fact the parent was fully mineralised. DOC was barely detected and the percentage biodegradation by DOC was not calculated, because the test item was not soluble in water at the test concentration (100 mg/L). The pH in all measured vessels was 7.3.

In the test solution containing sodium benzoate, the percentage biodegradation by BOD was 81 % on day 14. As per the OECD 301F Guideline, the test item can be assumed not to be toxic to the sludge microorganisms if the percentage biodegradation is >25 %. In the solution containing sodium benzoate + sludge (procedural control), the percentage biodegradation by BOD was 97 % on day 14. As per the OECD 301F Guideline, the test system can be assumed to be functioning correctly if the percentage biodegradation is => 60 %. In the procedural control, the biodegradation percentage was 97 % indicating that the test system performed as expected (=> 60 % value required by the validity criteria).

Whilst the 10-day window is not relevant for the test item as a UVCB substance, the percent biodegradation by BOD was 59 % and 60 % at the end of the 10-d window, therefore one of the replicates met the pass level of 60 % degradation in the 10 days immediately following attainment of 10 % biodegradation.

With an average 28 d biodegradation value of 91 % (BOD), the test item can be considered to be readily biodegradable under the conditions of this test.

All validity criteria for this test were fulfilled.

Description of key information

Readily biodegradable: 28-day biodegradation (based on BOD) = 91 %; OED 301F; Katagiri, K. (2019)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

A ready biodegradability test was performed in accordance with OECD Test Guideline 301F (Manometric Respirometry; 1992). All validity criteria for this test were fulfilled and the Japanese Guideline II: Method for Testing Biodegradability of Chemical Substances by Microorganisms (301F) stipulated in the "Testing Methods for New Chemical Substances".

The test item (concentration 100 mg/L) was added to two test vessels containing mineral medium inoculated with activated sludge (30 mg/L concentration of suspended solids) from a sewage treatment plant. Two inoculum blanks, a procedure blank and a toxicity control (reference substance: sodium benzoate) were run concurrently. A closed system oxygen consumption measurer was used to monitor BOD of the test solutions continuously during the incubation period of 28 days. At the end of the incubation period, for the test solutions and control blank, determination of DOC and pH was performed; as well as determination of the test item and presence of any degradation products by HPLC (as stipulated in the Japanese Gudeline).

The mean percentage biodegradation on day 28 was 91 % by BOD. Growth of the sludge was observed at the end of the incubation and no peaks corresponding to degradation products were detected on the HPLC chromatograms. DOC was barely detected (due to the substance being insoluble) and so the percentage biodegradation by DOC was not calculated. The pH in all measured vessels was 7.3.

In the test solution containing sodium benzoate, the percentage biodegradation by BOD was 81 % on day 14. As per the OECD 301F Guideline, the test item can be assumed not to be toxic to the sludge microorganisms if the percentage biodegradation is =>25 %. In the procedural control, the biodegradation percentage was 97 % indicating that the test system performed as expected (=> 60 % value required by the validity criteria).

With an average 28 d biodegradation value of 91 % (BOD), the test item can be considered to be readily biodegradable under the conditions of this test, because the pass level of 60 % biodegradadability was exceeded.