Cinnamomum zeylanicum, ext.

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Known constituent approach

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Cinnamon leaf oil is a Natural Complex Substance (NCS). With 94.5% of the constituents of Cinnamon leaf oil known, this NCS can be considered as a well defined NCS. Cinnamon leaf oil consists for 70 to 85% of eugenol and a variety of minor constituents. If the constituents of the NCS are readily biodegradable, the NCS itself is readily biodegradable as well.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Assessment of the Ready biodegradability of the known constituents; In the constituent approach (See Statement NCS protocol) for this purpose the NCS is considered as a mixture. For classification and labelling of the NCS, a summation approach is be applied as described for mixtures in the DPD and new Global Harmonization System (e.g. R53 or a GHS chronic classification if the sum of the percentages of constituents that are not rapidly degraded is ≥ 25%). From there it may be concluded, vice versa, that if ≥ 75% of sum of the percentage of the constituents is readily biodegradable, the NCS as a whole may be classified as readily biodegradable.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Remarks:

several guideline tests with different inocula were used

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

O2 consumption

Key result

Parameter:

% degradation (CO2 evolution)

Value:

> 60

Sampling time:

28 d

Remarks on result:

other: Cinnamic aldehyde, Alpha Phellandrene, Alpha-Pinene, Eugenol

Key result

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

28 d

Remarks on result:

other: Safrole, Linalool, Alpha Phellandrene, Benzyl benzoate, beta-carophyllene

Details on results:

Details for all constituents are summarised in window "Any other information on results" and in attached document

Assessment of the biodegradability of Cinnamon leaf oil:

Each of the identified constituents, totalling 94.5% of the typical composition, is readily biodegradable, see underlying data in table below.

Constituent	Typical %	Results of biodegradation assessment
Eugenol	75.3%	READILY BIODEGRADABLE EU-C.4-E (Closed Bottle Test): 82% (Haarmann & Reimer,1999) OECD 301B (Sealed CO2): 100.4% (Quest 1994 OECD 301F: 79% (7d) , 87% (28d) (Givaudan 1993)
Eugenyl acetate	3.3 %	Expected to be READILY BIODEGRADABLE Hydrolysis/cleavage to acetic acid and eugenol. Both transformation products are readily biodegradable
Benzyl benzoate	3.1 %	READILY BIODEGRADABLE - MITI (I) / OECD 301C: 28d - BOD: 90% Direct analysis: HPLC: 100% (MITI 1996: http://www.safe.nite.go.jp/sitemap/db_map_e.html)
b-carophyllene	3.7 %	READILY BIODEGRADABLE: - OECD301F - 75% (Givaudan, 2007)
Alpha-Pinene	1.24%	READILY BIODEGRADABLE: - OECD301B, sealed vessels: 62% (28d), 70% (42d) (Astra Zeneca, 2001, ECB PBT#84 http://ecb.jrc.ec.europa.eu/documents/PBT_EVALUATION/PBT_sum084_CAS_91770-80-8.pdf). - MITI (I) / OECD 301C: 28d - BOD: 91, 90, 95% Direct analysis: GC: 100, 100, 100% (MITI 2007: http://www.safe.nite.go.jp/sitemap/db_map_e.html)
Alpha-Phellandrene	1.33 %	READILY BIODEGRADABLE biodegradable based on structural analogy with terpinolene & d-limonene, see next rows Both have the same carbon skeleton; the only difference is the position of the double bonds. This is not expected to affect the sensitivity to biodegradation in a significant way
Structural analogue: D-limonene	--	Structural analogue of Alpha Phellandrene: READILY BIODEGRADABLE: - Headspace CO2: 71.4% (Quest 1993). The biodegradability of perfume ingredients in the sealed vessel test. - MITI (I) / OECD 301C, improved for volatile substance: Limonene 14d - BOD: 41, 81, 98% Direct analysis: GC: 100%; TOC 50, 73, 81% (MITI 1980 http://www.safe.nite.go.jp/sitemap/db_map_e.html) - Readily biodegradable (Misra, G.; Pavlostathis, S. G.; Perdue, E. M.; Araujo, R. Applied Microbiology and Biotechnology (1996), 45(6), 831-838.)
Structural analogue: Terpinolene	--	Structural analogue of Alpha Phellandrene: READILY BIODEGRADABLE - OECD 301B (sealed): 62% (Quest 1996): - OECD 301F: 51% (Givaudan, 1997) - OECD 302C: 80% (Givaudan, 1998) - Rapid primary degradation (Misra, G.; Pavlostathis, S. G.; Perdue, E. M.; Araujo, R. Applied Microbiology and Biotechnology (1996), 45(6), 831-838.)
Linalool	2.4 %	READILY BIODEGRADABLE: - OECD 301C: 80% (Givaudan 1991) - OECD Modified 301B, sealed CO2: 97.1% (QUEST 1994)                               - Readily biodegradable (Misra, G.; Pavlostathis, S. G.; Perdue, E. M.; Araujo, R. Applied Microbiology and Biotechnology (1996), 45(6), 831-838.)  - MITI (I) / OECD 301C: 28d - BOD: 90% Direct analysis: TOC 99% GC: 100%; (MITI 1996 http://www.safe.nite.go.jp/sitemap/db_map_e.html)
Safrole	0.9 %	READILY BIODEGRADABLE read-across from Eugenol (top of table) & Heliotropin (see next row) Supported by prediction from EPISuite/ BIOWIN v4.10 (March 2009): readily biodegradable in OECD 301C (MITI-test)
Structural analogue: Heliotropin CAS nr 120-57-0	--	Structural analogue of Safrole READILY BIODEGRADABLE - OECD 301F: 82% (Givaudan 1998) - OECD 301B (sealed CO2): 114% (Quest 1993)
Cinnamic aldehyde	1.3%	READILY BIODEGRADABLE OECD 301B - CO2 evolution (Sealed vessel): 89% (7d), 94% (14d), 100% (21d, 27d, 28d) Source: Robust Summaries for Cinnamyl Derivatives, FFHPVC Aromatic Consortium Registration Number, 201 – 15843B (OPPT 25 March 2005) http://www.epa.gov/hpv/pubs/summaries/cinna/c12912rr.pdf comm. Haarmann and Reimer (2001) to RIFM
Cinnamyl acetate	1.9%	Expected to be READILY BIODEGRADABLE According to the UM-BBD Pathway predictions (http://umbbd.ethz.ch/predict/),the ester bond will be broken (bt0024) and cinnamic alcohol will be formed which is readily biodegradable (see above) EPIWIN predictions for c1ccccc1C=CCOC(=O)C: READILY BIODEGRADABLE BIOWIN2 0.9659 BIOWIN3 3.8273 BIOWIN6 0.7293
	94.5%	Fraction of NCS readily biodegradable or expected to be readily biodegradable

 The complete asssessment is added attached to this study record. Studies mentioned by Company Name are included in the RIFM database.

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

Cinnamon leaf oil is considered as readily biodegradable.

Executive summary:

Cinnamon leaf oil is a Natural Complex Substance (NCS) for which > 90% of the constituents is identified. The main constituent in Cinnamon leaf oil is Eugenol (range 70-85% of the composition). As no valid empirical results from a test for the ready biodegradation of Cinnamon leaf oil are available, the assessment is based on its constituents (maximum in composition at or above 1%). The assumption is that if the constituents of the NCS are readily biodegradable, the NCS itself is readily biodegradable as well.

With the exception of 3 minor constituents, for all constituents the available data are obtained in tests on the ready biodegradability. For the 3 minor constituents the ready biodegradability is based on structurally related substances.

 

The constituents, comprising 94.5% of the typical composition of this NCS, are in the category of ready biodegradable substances. Therefore, Cinnamon leaf oil is considered readily biodegradable.

 

 

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-Mar-2022 - 25-Apr-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Version / remarks:

1994

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

yes

Remarks:

Ammonium chloride present in the mineral salt medium was omitted because it may be converted to nitrate which leads to an oxygen consumption not related to the biodegradation of the test substance.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Obtained from sponsor
- Test Substance name: Cinnamon Leaf Oil
- Other name: C03319 CINNAMON LEAF EO SRI-LANKA
- Batch no.: 5030062511
- CAS no.: 84649-98-9
- Appearance: brown, yellow liquid
- Expiry date: 02-Apr-2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool, dry place with adequate ventilation
- Chemical stability: Stable under normal temperature conditions and recommended use

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum: River water was sampled from the Rhine near Heveadorp, The Netherlands (21-Mar-2022). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.

- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The Colony forming units (CFU) in the particle free and preconditioned river water was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned river water used in the closed bottles was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.1 106 CFU/L.

Reference: *Ginkel CG van and Stroo CA (1992) Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotoxicol Environ Saf 24 319-327.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Deionized water:
Deionized water containing <1.0 mg/L of organic carbon was prepared in a water purification system.

Nutrients, stocks and administration:
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.50 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification that is not related to the biodegradation of the test substance.
Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and the content was mixed vigorously until there were no more lumps in the silica gel, indicating homogeneous distribution of the test substance. Subsequently, 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with the river water which was spiked with the nutrients and closed. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.

Test procedure:
Use was made of 10 bottles containing only river water with nutrients, 10 bottles containing river water with nutrients and silica gel (0.2 g silica gel / bottle), 10 bottles containing river water with nutrients and the silica gel spiked with the test substance, 6 bottles containing river water with nutrients and sodium acetate. The concentrations of the test substance and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of the control, the control with silica gel and the test substance were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. Two duplicate bottles of sodium acetate were withdrawn for analyses of the dissolved oxygen concentration at day 7 and 14.

Test conditions:
At the start of the test the pH of the media in the duplicate bottles was 7.8, 8.1, 8.0 and 8.1 in the control bottles, the test bottles, the silica gel control bottles and the reference bottles, respectively. The pH of the medium in the duplicate reference bottles measured at day 14 was 7.9. The pH of the medium in the duplicate bottles at day 28 was 8.1, 7.7 and 7.7 for the control, the test substance, and the control with silica gel bottles, respectively. The temperature ranged from 21.7 to 23.0°C which is within the prescribed temperature range of 20 to 24°C.

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.8% Batch/lot number: BCCD4856

Test performance:

Validity of the test:
The validity of the test is demonstrated by an endogenous respiration of 1.45 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 76%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Toxicity:
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test is optional in the OECD guideline and was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition can be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance (higher oxygen concentration in bottles with test substance compared to the concentration in the control bottles). Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Key result

Parameter:

% degradation (O2 consumption)

Value:

96

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

73

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

0 d

Details on results:

The substance was biodegraded by 96% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation, therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds. Hence this substance should be classified as readily biodegradable.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 76%.

Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Mc	Ma	Mcs	Mt
0	8.7	8.7	8.7	8.8
	8.7	8.7	8.7	8.7
Mean (M)	8.70	8.70	8.70	8.75
7	7.8	4.1	8.3	4.7
	7.8	4.1	8.1	5.0
Mean (M)	7.80	4.10	8.20	4.85
14	7.5	3.5	7.7	3.8
	7.5	3.5	7.7	4.5
Mean (M)	7.50	3.50	7.70	4.15
21	7.3		7.6	4.1
	7.3		7.6	3.7
Mean (M)	7.30		7.60	3.90
28	7.3		7.4	2.8
	7.2		7.4	2.6
Mean (M)	7.25		7.40	2.70

Mc       River water with nutrients

M_a       River water with nutrients and sodium acetate (6.7 mg/L).

M_cs      River water with nutrients and silica gel

M_t       River water with nutrients, test substance (2 mg/L) and silica gel

 

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance and sodium acetate (BOD/ThOD_NH3) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	-0.05*	0.00	0	0
7	3.35	3.70	69	71
14	3.55	4.00	73	76
21	3.70		76
28	4.70		96

* The small oxygen difference between the test substance and silica gel control bottles at the start of the test (day 0) is a result of the inaccuracy of the oxygen measurements and should be interpreted as 0% biodegradation.

 

 

 

 

Validity criteria fulfilled:

yes

Remarks:

See "test performance" for details

Interpretation of results:

readily biodegradable

Conclusions:

Cinnamon leaf oil was biodegraded by 96% at day 28 in the Closed Bottle test and should be classified as readily biodegradable.

Executive summary:

The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with GLP. Cinnamon leaf oil  oil did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Validity criteria were met. The test item was biodegraded by 96% at day 28. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation thereby fulfilling the time window criterion for ready biodegradable substances. The test item should therefore be classified as readily biodegradable.

Description of key information

Cinnamon leaf oil is a Natural Complex Substance (NCS) with eugenol as the main constituent, ranging from 70 - 85%. In a Closed Bottle test (OECD TG 301D) Cinnamon leaf oil was biodegraded by 96% at day 28. In addition all known constituents comprising 94.5% of the typical composition of this NCS are in the category of ready biodegradable substances. Therefore, Cinnamon leaf oil is considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater