D,L- Menthol / D,L-Isomenthol

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:

23 January 2018 to 12 February 2018

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge freshly obtained from a wastewater treatment plant which treats predominantly domestic sewage.
- Pre-treatment: None.
- Preparation of inoculum for exposure: The activated sludge was washed three times via the processes of centrifugation, decantation of the liquid phase and resuspension in tap water and mineral medium.
- Concentration of sludge: The concentration of the sludge was 30 mg/L

Duration of test (contact time):

18 d

Initial conc.:

178 mg/L

Based on:

ThOD

Initial conc.:

60 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solution 1: 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4 x 2H2O, 0.5 g/L NH4Cl
Stock solution 2: 36.40 g/L CaCl2 x 2H2O
Stock solution 3: 22.50 g/L MgSO4 X 7H2O
Stock solution 4: 0.25 g/L FeCl3 x 6H2O, stabilised with a drop of concentrated hydrochloric acid per litre.
The mineral medium contained 10 mL of stock solution 1 and 1 mL each from stock solutions 2-4; which was then mixed with ultrapure water and made up to a volume of 1L. Diluted hydrochloric acid solution was used to make a pH adjustment from 7.8 to 7.4.

- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: 21° C
- pH: 7.3 – 7.7
- pH adjusted: The pH of the mineral medium was adjusted from 7.8 to exactly 7.4 before the start of the test.
- CEC (meq/100 g): Not applicable
- Aeration of dilution water: Test vessels were incubated under continuous stirring
- Suspended solids concentration: 30 mg dry material per litre
- Continuous darkness: The test vessels were incubated under diffused lighting

TEST SYSTEM
- Culturing apparatus: Laboratory glass bottles GL45 with a nominal volume of 500 mL were
used. The test vessels contained 164 mL of test suspension, a suitable volume to assure sufficient oxygen in the headspace. The prepared test vessels were air-tight closed with the OxiTop measuring heads.
- Number of culture flasks: Two test vessels were prepared for the test item, inoculum control and procedure control and one test vessel for the toxicity control
- Method used to create aerobic conditions: aeration
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: OxiTop OC110 controller was used to convert collected pressure values to BOD values.
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Sodium Hydroxide pellets were used to absorb CO2.


SAMPLING
- Sampling frequency: The pressure reduction was continuously detected by the OxiTop pressure
measuring heads and recorded every three hours.
- Sampling method: See above
- Sterility check if applicable: Not applicable
- Sample storage before analysis: Not applicable
- Other: Not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes
- Other: Procedure control also included (reference item sodium benzoate)

STATISTICAL METHODS: Percentage biodegradation was calculated based on BOD and ThOD (see below)

Reference substance:

other: sodium benzoate

Test performance:

A reduced test item concentration of 60 mg test item/L was used to prevent possible toxicity to the inoculum. Toxicity, reference and inoculum controls were performed during the test. All controls fulfilled the validity criteria therefore confirming that the test system worked.

Key result

Parameter:

% degradation (O2 consumption)

Value:

79

Sampling time:

18 d

Details on results:

-The test was ended ahead of the scheduled 28-day exposure. This was because a plateau in the biodegradation curve was reached after 18 days.
- The biodegradation values calculated from the measurements performed during the test period revealed 79 % biodegradation of the test item (mean of two replicates. Thus, the criterion for ready biodegradability (at least 60% biodegradation within the 10-day window) was met.
- Toxicity control: In the toxicity control more than 25% biodegradation occurred within 14 days (68%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity.

Results with reference substance:

The reference substance sodium benzoate reached 88 % degradation within 14 days, therefore the suitability of the activated sludge was confirmed.

Table 1- Biodegradation percentages of test item and reference item during incubation period

	Biodegradation percentage (%)
Time (days)	Test item		Procedure control		Toxicity control
	Replicate 1	Replicate 2	Replicate 1	Replicate 2	Replicate 1
0	0	0	0	0	0
1	-1	-1	27	30	15
2	-2	-1	50	53	24
3	0	-1	66	68	32
4	5	3	73	75	35
5	30	36	77	79	37
6	47	50	78	81	39
7	62	54	81	84	45
8	67	55	82	86	52
9	70	55	83	87	56
10	72	56	84	87	61
11	75	59	85	89	64
12	76	63	85	89	66
13	78	70	86	90	67
14	77	71	85	90	68
15	80	75	86	92	70
16	81	77	87	93	71
17	79	75	87	92	73
18	80	77	88	92	74
Mean (day 18)	79		90		Not applicable

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In conclusion, the test item was readily biodegradable under the conditions of the test (79 % mean biodegradation after 18 days based on the Manometric Respirometry Test).

Executive summary:

The test item was investigated for its ready biodegradability in a Manometric Respirometry Test according to OECD 301 F. In accordance with the test guidelines the test was ended after 18 days, ahead of the scheduled 28 exposure days, since the biodegradation curve of the test item had reached a plateau. To avoid a possible toxicity to the inoculum, the test was performed with a reduced test item concentration, i.e. 60 mg test item/L giving approximately 178 mg Theoretical Oxygen Demand/L. The reference substance achieved 88 % degradation within 14 days and the toxicity control showed 68 % degradation after 14 days, therefore meeting the required test validity criteria. Degradation of 79 % was determined for the test item within the test period of 18 days. In accordance with the test guidelines the test was ended after 18 days, ahead of the scheduled 28 exposure days, since the biodegradation curve of the test item had reached a plateau. Consequently, the test item was found to be readily biodegradable under the test conditions. 

Description of key information

In conclusion, the test item was readily biodegradable under the conditions of the test (79 % mean biodegradation after 18 days based on the Manometric Respirometry Test).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The test item was investigated for its ready biodegradability in a Manometric Respirometry Test according to OECD 301 F. In accordance with the test guidelines the test was ended after 18 days, ahead of the scheduled 28 exposure days, since the biodegradation curve of the test item had reached a plateau. To avoid a possible toxicity to the inoculum, the test was performed with a reduced test item concentration, i.e. 60 mg test item/L giving approximately 178 mg Theoretical Oxygen Demand/L. The reference substance achieved 88 % degradation within 14 days and the toxicity control showed 68 % degradation after 14 days, therefore meeting the required test validity criteria. Degradation of 79 % was determined for the test item within the test period of 18 days. In accordance with the test guidelines the test was ended after 18 days, ahead of the scheduled 28 exposure days, since the biodegradation curve of the test item had reached a plateau. Consequently, the test item was found to be readily biodegradable under the test conditions.