Menthol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2002-10-11 till 2003-01-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 218

RADIOLABELLING INFORMATION (if applicable): No radiolabelling

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: No data

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic wastewater. The activated sludge was transported in a plastic bottle and aerated until use.
Concentration of inoculum: 5 ml/L

Duration of test (contact time):

28 d

Initial conc.:

0.84 mg/L

Based on:

test mat.

Initial conc.:

2.01 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Reference substance:

other: sodium acetate

Parameter:

% degradation (O2 consumption)

Value:

92

Sampling time:

28 d

Remarks on result:

other: Test conc. 0.84 mg/l

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Test conc. 2.01 mg/l

Parameter:

COD

Value:

2.5 other: mg O2/L

Parameter:

COD

Value:

5.97 other: mg O2/L

Results with reference substance:

The results show the expected degradation of sodium acetate (> 60 % within 14 days). The calculated Biochemical Oxygen Demand (BOD) of sodium acetate after 7 and 14 days was 0.59 and 0.70 mg Oxygen mg/L respectively, which corresponds with its Theoretical Oxygen Demand (ThOD) of 0.68 mg Oxygen mg/L.

Biodegradation of D-Menthol expressed as the BOD (mgO2 mg/l) and as percentage of its ThOD.

Test concentration: 0.84 mg/L

Time (days)	BOD (mg O2/mg)	Biodegradation (%)
7	1.89	64
14	2.72	92
21	2.67	90
28	2.74	92

Biodegradation of D-Menthol expressed as the BOD (mgO2 mg/l) and as percentage of its ThOD.

Test concentration: 2.01 mg/L

Time (days)	BOD (mg O2/mg)	Biodegradation (%)
7	1.82	61
14	2.13	72
21	2.25	76
28	2.26	76

Validity criteria fulfilled:

yes

Remarks:

reference compound (sodium acetate) > 60 % within 14 days. -In the toxicity control degradation rates > 25 % within 14 days

Interpretation of results:

readily biodegradable

Conclusions:

Within 28 days, a degradation rate of 92 % and 76 % respectively was determined for D-Menthol.

Executive summary:

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed and after 28 days 92 % of D-Menthol (CAS no. 15356-60-2) was degraded in a test with an initial concentration of 0.84 mg/l. 76 % of D-Menthol was degraded in a test with an initial concentration of 2.01 mg/l. Therefore, D-Menthol is considered being readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See 'Attached justification' document: Justification for a read-across approach from L-Menthol (CAS No 2216-51-5, EC No 218-690-9) [Source] to D/L-Menthol (CAS No 89-78-1, EC No 201-939-0) [Target].

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (O2 consumption)

Value:

92

Sampling time:

28 d

Remarks on result:

other: Test conc. 0.84 mg/l

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Test conc. 2.01 mg/l

Parameter:

COD

Value:

2.5 other: mg O2/L

Parameter:

COD

Value:

5.97 other: mg O2/L

Results with reference substance:

The results show the expected degradation of sodium acetate (> 60 % within 14 days). The calculated Biochemical Oxygen Demand (BOD) of sodium acetate after 7 and 14 days was 0.59 and 0.70 mg Oxygen mg/L respectively, which corresponds with its Theoretical Oxygen Demand (ThOD) of 0.68 mg Oxygen mg/L.

Biodegradation of D-Menthol expressed as the BOD (mgO2 mg/l) and as percentage of its ThOD.

Test concentration: 0.84 mg/L

Time (days)	BOD (mg O2/mg)	Biodegradation (%)
7	1.89	64
14	2.72	92
21	2.67	90
28	2.74	92

Biodegradation of D-Menthol expressed as the BOD (mgO2 mg/l) and as percentage of its ThOD.

Test concentration: 2.01 mg/L

Time (days)	BOD (mg O2/mg)	Biodegradation (%)
7	1.82	61
14	2.13	72
21	2.25	76
28	2.26	76

Validity criteria fulfilled:

yes

Remarks:

reference compound (sodium acetate) > 60 % within 14 days. -In the toxicity control degradation rates > 25 % within 14 days

Interpretation of results:

readily biodegradable

Conclusions:

Within 28 days, a degradation rate of 92 % and 76 % respectively was determined for D-Menthol.

Executive summary:

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed and after 28 days 92 % of D-Menthol (CAS no. 15356-60-2) was degraded in a test with an initial concentration of 0.84 mg/L. 76 % of D-Menthol was degraded in a test with an initial concentration of 2.01 mg/L. Therefore, D-Menthol is considered being readily biodegradable.

The OECD SIDS report on Menthol concludes on page 4 that “the menthols can be considered as a category because of their similarity in physico-chemical, toxicological, ecotoxicological and environmental fate properties” which could be an indicator to justify the application of the read-across approach.

In the OECD SIDS report (2003) biodegradability is given in the range of 76 - 92 % after 28 days for D-Menthol (CAS no. 15356-60-2) and 79 - 92 % after 28 days for L-Menthol (CAS no. 2216-51-5) determined according to OECD 301D tests. These two closed-bottle tests with D-Menthol and L-Menthol show clearly that both isomers are readily biodegradable. As DL-Menthol is mostly a racemic mixture of D- and L-Menthol, it seems likely that DL-Menthol is also readily biodegradable. The study on D-Menthol, showing the lower biodegradation rate of 76 - 92 %, was was used for read-across.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Principles of method if other than guideline:

batch system (comparable to Zahn-Wellens Test OECD TG 302B)

GLP compliance:

no

Specific details on test material used for the study:

OTHER SPECIFICS: Name of the test substance as cited in the report: Menthol

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from a sewage plant, activated sludge, adapted for 20 days.
- Laboratory culture: cultivated in a 1000 mL cilinder
- Method of cultivation: The mixture is aerated with pressure air. Every day 200 mL of the mixture is driven off so that sludge age is 5 days.
- Preparation of inoculum for exposure: After sedimentation ca. 600 mL of the liquid phase is driven off. The residue (200 ml of the thickened
activated sludge) is diluted with tap water to the volume of ca. 800 mL and 600 mg/L of starch or glucose, 600 mg/L of pepetone, 25 mL of a phosphate buffer and the solution of the tested compund are added.
- Pretreatment: The mixture in the cilinder (see above) is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is
repeated, and the concen-tration of the test substance is gradually increased so that after 20 days of adaptation it reaches the value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L (dry matter)

Initial conc.:

200 mg/L

Based on:

COD

Details on study design:

Pretreatment: The mixture in the cilinder made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure repeated, and the concentration of the test substance gradually increased so that after 20 days of adaptation it reached the value of 200 mg/L COD (chemical oxygen demand).
The initial value of COD or organic carbon of the liquid phase was determined.
Samples (ca. 50-80 mL), filtered or centrifugated before analysis, taken at suitable intervals. The decrease of the test substance in the liquid phase is evaluated by determining COD or organic carbon. The results compared with those of a blank test and standard compound decomposition.
With the degree of degradation also the average specific rate of degradation determined, expressed in terms of mg COD (or organic carbon) removed by a gramme of dry matter of the activated sludge per hour. The experiment is carried out till there is no decrease of COD. After that time the total percentage of COD removed and the rate of degradation are evaluated.

Parameter:

other: COD

Value:

95.1

Sampling time:

5 d

Details on results:

The measured COD in samples was compared with those of the blank test and standard compound decomposition.
The substance tested can be considered to be biologically decomposable, since 90 % of the initial COD of the substance is removed in 120 h of
incubation under the experimental conditions. Inition degradation rate: 17.7 mg COD g/h

A test on inherent biodegradability was conducted by Pitter (1976). The test design is comparable to the Zahn-Wellens-test (OECD TG 302 B). The test substance Menthol in a concentration of 200 mg/L COD was the sole source of carbon. Activated sludge from a sewage treatment plant adapted for 20 days to Menthol was used as inoculum in a concentration of 100 mg/L dry matter. Based on COD measurement, a removal of 94.6 % within 5 days was obtained.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Within 5 days a degradation of 95.1 % was determined equivalent or similar to the OECD Guideline 302 B.

Executive summary:

A test on inherent biodegradability was conducted by Pitter (1976). The test design is comparable to the Zahn-Wellens-test (OECD TG 302 B). The test substance DL-Menthol in a concentration of 200 mg/L COD (chemical oxygen demand) was the sole source of carbon. Activated sludge from a sewage treatment plant adapted for 20 days to Menthol was used as inoculum in a concentration of 100 mg/L dry matter.

95.1 % of DL-Menthol was degraded within five days. Therefore, DL-Menthol is considered being inherently biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2002-10-11 till 2003-01-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-Batch number: 132

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic wastewater. The activated sludge was transported in a plastic bottle and aerated until use.
Concentration of inoculum: 5 ml/L
Total volume inoculated mineral medium: 50 L

Duration of test (contact time):

28 d

Initial conc.:

0.84 mg/L

Based on:

test mat.

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Preparation of the bioderadation test bottles:
A stock solution was made by dissolving 0.2002 g of test substance Menthol L Dist. in one litre of ultrapure water. Suitable volumes of this stock solution were added to inoculated medium to obtain test substance concentrations of 0.84 and 2.00 mg/L. This series was completed with an inoculum blank, containing only inoculated mineral medium.

Preparation of the activity control:
The final test concentration of sodium acetate was prepared by dissolving 0.3998 g of the reference substance in 100 ml of ultrapure water. From this stock solution a dilution was male in inoculated mineral medium to give a final nominal test concentration of 4.00 ing/L sodium acetate.

Preparation of the toxicity control:
Dilutions of the test substance and the reference substance stock solutions were prepared in inoculated mineral medium. The toxicity control contained 2.00 mg/L Menthol L Dist. and 4.00 mg/L sodium acetate.

lncubation and rneasurements:
The filled BOD bottles were closed and incubated at about 20°C in the dark after measurernent of the oxygen concentration in one bottle of each series. To prevent leakage of gasses out of the BOD bottles, they were incubated up side down standing an their plug. The oxygen concentrations were measured using an oxygen electrode. Thereafter a separate set of four bottles was sacrificed for oxygen measurements at each time-point. The temperature in one bottle of each treatment was measured at each time-point indicated above.
The pH in one bottle of each treatment was measured at the start of the test and after 28 days of incubation, except for the control bottles with sodium acetate and the toxicity controls with sodium acetate and test substance that were measured after 14 days of incubation.

Reference substance:

other: sodium acetate

Parameter:

% degradation (O2 consumption)

Value:

92

Sampling time:

28 d

Remarks on result:

other: Test conc. 0.84 mg/l

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Test conc. 2.00 mg/l

Parameter:

BOD5

Value:

2.35 other: mg O2/mg test material

Parameter:

BOD5

Value:

2.74 other: mg O2/mg test material

Results with reference substance:

The results show the expected degradation of sodium acetate (> 60 % within 14 days). The calculated Biochemical Oxygen Demand (BOD) of sodium acetate after 7 and 14 days was 0.59 and 0.70 mg Oxygen mg/L respectively, which corresponds with its Theoretical Oxygen Demand (ThOD) of 0.68 mg Oxygen mg/L.

Biodegradation of L-Menthol expressed as the BOD (mgO2 mg) and as percentage of its ThOD.

Test concentration: 0.84 mg/L

Time (days)	BOD (mg O2/mg test material)	Biodegradation (%)
7	1.90	64
14	2.66	90
21	2.77	93
28	2.74	92

Biodegradation of L-Menthol expressed as the BOD (mgO2 mg) and as percentage of its ThOD.

Test concentration: 2.00 mg/L

Time (days)	BOD (mg O2/mg test material)	Biodegradation (%)
7	1.90	64
14	2.27	76
21	2.28	77
28	2.35	79

Validity criteria fulfilled:

yes

Remarks:

Reference compound (sodium acetate) showed > 60 % degradation within 14 days. Degradation rates > 25 % within 14 days in the toxicity control. < 20 % differences of replicate values. Oxygen depletion in the inoculum blank was 1.16 mg O2/L.

Interpretation of results:

readily biodegradable

Conclusions:

Within 28 days, a degradation rate of 93% (at a test concentration of 0.84 mg/L) and 76 % (at a test concentration of 2.00 mg/L) respectively was determined for L-Menthol.

Executive summary:

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed. After 28 days 93 % of L-Menthol (CAS no. 2216 -51 -5) degraded in a test with an initial concentration of 0.84 mg/l. 79 % of L-Menthol degraded in a test with an initial concentration of 2.00 mg/l. Therefore, L-Menthol is considered being readily biodegradable.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See 'Attached justification' document: Justification for a read-across approach from L-Menthol (CAS No 2216-51-5, EC No 218-690-9) [Source] to D/L-Menthol (CAS No 89-78-1, EC No 201-939-0) [Target].

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Parameter:

% degradation (O2 consumption)

Value:

92

Sampling time:

28 d

Remarks on result:

other: Test conc. 0.84 mg/l

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Test conc. 2.00 mg/l

Parameter:

BOD5

Value:

2.35 other: mg O2/mg test material

Parameter:

BOD5

Value:

2.74 other: mg O2/mg test material

Results with reference substance:

The results show the expected degradation of sodium acetate (> 60 % within 14 days). The calculated Biochemical Oxygen Demand (BOD) of sodium acetate after 7 and 14 days was 0.59 and 0.70 mg Oxygen mg/L respectively, which corresponds with its Theoretical Oxygen Demand (ThOD) of 0.68 mg Oxygen mg/L.

Biodegradation of L-Menthol expressed as the BOD (mgO2 mg) and as percentage of its ThOD.

Test concentration: 0.84 mg/L

Time (days)	BOD (mg O2/mg test material)	Biodegradation (%)
7	1.90	64
14	2.66	90
21	2.77	93
28	2.74	92

Biodegradation of L-Menthol expressed as the BOD (mgO2 mg) and as percentage of its ThOD.

Test concentration: 2.00 mg/L

Time (days)	BOD (mg O2/mg test material)	Biodegradation (%)
7	1.90	64
14	2.27	76
21	2.28	77
28	2.35	79

Validity criteria fulfilled:

yes

Remarks:

Reference compound (sodium acetate) showed > 60 % degradation within 14 days. Degradation rates > 25 % within 14 days in the toxicity control. < 20 % differences of replicate values. Oxygen depletion in the inoculum blank was 1.16 mg O2/L.

Interpretation of results:

readily biodegradable

Conclusions:

Within 28 days, a degradation rate of 93% (at a test concentration of 0.84 mg/L) and 76 % (at a test concentration of 2.00 mg/L) respectively was determined for L-Menthol.

Executive summary:

A read-across approach was used used for data gap filling of D/L-Menthol (CAS No 89-78-1) for the endpoint ´Biodegradation in water: screening test` by using the data of L-Menthol (CAS No 2216-51-5). As L Menthol is a natural product, it is enantiomer pure (enantiomeric purity ≥ 99 %) and therefore an ideal source substance for a read across approach. Based on the similar chemical structure, source and target substance have comparable properties. L-Menthol and D/L Menthol (racemic mixture of the D- and the L-isomer) exhibit similar structures, as D-Menthol and L-Menthol are two stereoisomers that are mirror images of each other. It is experimentally verified that L-Menthol and D/L-Menthol have nearly identical behavior in physico-chemical and (eco)toxicological studies. Therefore, there should be almost no differentiation between effects of target and source substance.

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed. After 28 days 93 % of L-Menthol (CAS no. 2216 -51 -5) degraded in a test with an initial concentration of 0.84 mg/l. 79 % of L-Menthol degraded in a test with an initial concentration of 2.00 mg/l. Therefore, L-Menthol is considered being readily biodegradable.

Description of key information

Ready biodegradability:

Read-across

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed and after 21 days a maximum percentage of 93 % biodegradation of L-Menthol (CAS no. 2216 -51 -5) was reached in a test with an initial concentration of 0.84 mg/L. 79 % of D-Menthol was degraded in a test with an initial concentration of 2.00 mg/L aftre 28 days. Therefore, L-Menthol is considered being readily biodegradable.

A closed bottle test comparable to OECD TG 301 D under aerobic conditions was performed and after 28 days 92 % of D-Menthol (CAS no. 15356-60-2) was degraded in a test with an initial concentration of 0.84 mg/L. 76 % of D-Menthol was degraded in a test with an initial concentration of 2.01 mg/L. Therefore, D-Menthol is considered being readily biodegradable.

Inherent biodegradability:
A test on inherent biodegradation comparable to OECD TG 302 B was performed with a duration of 5 days (Pitter, 1976). The test substance Menthol in a concentration of 200 mg/L COD (chemical oxygen demand) was the sole source of carbon. Activated sludge from a sewage treatment plant adapted for 20 days to Menthol was used as inoculum in a concentration of 100 mg/L dry matter.

95.1 % of DL-Menthol was degraded during the test period. Therefore, DL-Menthol is considered being inherent biodegradable.

The OECD SIDS report on Menthol concludes on page 4 that “the menthols can be considered as a category because of their similarity in physico-chemical, toxicological, ecotoxicological and environmental fate properties” which could be an indicator to justify the application of the read-across approach.

As both studies show biodegradability for isomers of Menthol it can be concluded, that other isomers of Menthol could also be considered as biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Ready biodegradation:

Another test was conducted in accordance with "Biodegradation test of chemical substance by microorganisms etc." stipulated in the Order Prescribing the Items of the Test Relating to the New Chemical Substance (1974, Order of the Prime Minister, the Minister of Health and Welfare, the Minister of International Trade and Industry No. 1). This guideline corresponds to "301C, Ready Biodegradability: Modified MITI Test (I)" stipulated in the OECD Guidelines for Testing of Chemicals (1981).

As no biodegradation is found within 28 d the substance would be considered as "not readily biodegradable". However, as other studies imply a ready biodegradability of Menthol, the biodegradation seems to be affected by the toxicity of the substance at the concentration tested. Thus this study has to be classified as invalid.