Amyris balsamifera, ext.

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-04-2012 to 15-05-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amyris balsamifera, ext.

EC Number:

291-076-6

EC Name:

Amyris balsamifera, ext.

Cas Number:

90320-49-3

Molecular formula:

Not applicable due to the UVCB nature of the substance.

IUPAC Name:

Essential oil of Amyris obtained from the wood of Amyris balsamifera (Rutaceae) trees by steam distillation.

Test material form:

liquid: viscous

Remarks:

Pale yellow slightly viscous liquid

Details on test material:

Name of test material as cited in study report: Amyris oil
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

Specific details on test material used for the study:

Appearance: yellow to pale yellow liquid
lot no.: VE00195303
purity: GC conform
expiration date: July 1, 2013
Elemental analysis: 81.99% C, 12.01% H

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switserland) was used.
- Pretreatment: The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
- Concentration of sludge:
*Dry weight of suspended solids: 7.93 g/L, diluted to 1.53 g/L.
*To obtain a concentration of 30 mg/L (dry weight) in 103 mL total volume, 2.00 mL of sludge was added (inoculum).
*To obtain a concentration of 30 mg/L (dry weight) in 255 mL total volume, 5.00 mL of sludge was added (inoculum)
The dry weight of suspended solids is determined by taking two 50mL samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110 °C for two hours and weighing the residue.

Duration of test (contact time):

35 d

Initial test substance concentration

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solution A:
KH2PO4: 8.5 g
K2HPO4: 21.75 g
NA2HPO4 * 2 H2O: 33.4 g
NH4Cl: 0.5 g
Dissolved in water and made up to 1 litre.

Stock solution B:
CaCl2: 27.5 g
Dissolved in water and made up to 1 litre.

Stock solution C:
MgSO4 * 7 H2O: 22.5 g
Dissolved in water and made up to 1 litre.

Stock solution D:
FeCl3 * 6H2O: 0.25 g
HCL Conc. one drop
Dissolved in water and made up to 1 litre.

- Deionised water: Containing less than 10 mg/L dissolved organic carbon.
- Mineral medium: Prepared by mmixing 50 mL of solution A and 2000 mL deionised water, adding 5 mL of each of the solutions B, C, and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium.
- Test temperature: 22.1 - 22.6 °C
- pH: initial pH 7.6, final pH 7.49 - 8.21
- pH adjusted: if necessary


TEST SYSTEM
- Number of culture flasks/concentration: 2
- Test substance samples (7.65 mg, corresponding to 30.0 mg/L in 255 mL test medium) were weighed in small aluminium boats and added directly to the test flasks of the Oxitop whereas reference the substance sodium benzoate was added as 1.00 mL of a 10.2 g/L solution in mineral medium, to give a total volume of 103 mL.
Flasks were filled with 250 mL of mineral medium (flasks containing the reference substance: 100 mL). Samples of test or reference substance were added. Then suspended sludge diluted to a concentration of 1.53 g/L dry matter was added. Except when the test substance has an acid or alkaline character, the pH of each flask was not measured but assumed the same as the mineral medium, in order to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium more than 0.1 unit. Two sodium hydroxide pellets were placed in the quivers on top of the bottle, and the flasks were closed tightly with the measuring heads. The flasks were allowed to equilibrate to the test temperature.
- Measuring equipment: - Apparatus: The respirometer used during this study is an Oxitop Control System, made by Wissenschaftlich-Technische werkstatten (WTW), Weilheim, Germany.
- Details of trap for CO2 and volatile organics if used: Sodium hydroxide pellets

SAMPLING
- Sampling frequency: Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring were checked. At the end of the test period, the pH od each flask was measured again. Results of day 1, 3, 4, 14, 21, 28, and 35 were reported in the study report.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity ≥ 99%, Art. No. 71300 Fluka Buchs Switzerland

Results and discussion

Test performance:

Amyris Oil did not inhibit the intrinsic respiration of the inoculum at the test concentration. Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days.The difference between replicates is not more than 20%. The test was considered valid.

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

-2

Sampling time:

1 d

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

3 d

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

18

Sampling time:

4 d

% Degradation 4

Parameter:

% degradation (O2 consumption)

Value:

51

Sampling time:

14 d

% Degradation 5

Parameter:

% degradation (O2 consumption)

Value:

58

Sampling time:

21 d

% Degradation 6

Key result

Parameter:

% degradation (O2 consumption)

Value:

62

Sampling time:

28 d

% Degradation 7

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

35 d

Details on results:

Amyris Oil Dominican Republic undergoes 62% biodegradation after 28 days (63% after 35 days). The 10-day window criterion is not fulfilled (18% biodegradation on day 4 and 51% on day 14). However, Amyris Oil is a natural complex substance consisting of structurally similar chemicals which are expected to have similar degradation potential. The degradation curve will therefore be the sum of multiple growth curves. The 10-day window criterion was developed on the assumption that a test substance (i.e. monoconstituent substance) is degraded according to a "standard" growth curve in ready biodegradability test. The 10-day time-window should therefore be ignored as a pass/fail criterion for Amyris oil. Thus Amyris Oil Dominican Republic should be regarded as readily biodegradable.

BOD5 / COD results

Results with reference substance:

Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days.

Any other information on results incl. tables

O2 uptake (mg O2/L adjusted to nominal concentrations)

Amyris Oil	Days		1	3	4	14	21	28	35
O2 uptake of sludge (inoculum blank)		B1	8.1	17.5	20.2	35.0	40.4	43.1	47.1
		B2	8.1	17.5	20.2	32.3	37.7	41.7	43.1
	Mean	B	8.1	17.5	20.2	33.7	39.1	42.4	45.1
O2 uptake of test Subst. + sludge		C1	5.4	21.5	35.0	79.3	91.4	96.8	101.9
		C2	6.7	21.5	39.1	82.4	93.2	101.3	104.3
O2 uptake of test substance		C1-B	-2.7	4.0	14.8	45.7	52.4	54.4	56.8
		C2-B	-1.4	4.0	18.9	48.7	54.1	58.9	59.2
% biodegradation of test substance		D1	-3	4	16	50	57	59	62
		D2	-2	4	21	53	59	64	64
	mean	D	-2	4	18	51	58	62	63

Calculations

B1, B2, C1, C2, A1, A2, E1: experimental O2 uptake values.

B = (B1 + B2)/2

D1 = 100 * (C1-B) / ThOD * [S]

D2 = 100 * (C2 – B)/ ThOD * [S]

D = (D1 + D2)/2

[S] = initial test substance concentration (mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See "test performance" for details

Interpretation of results:

readily biodegradable

Remarks:

Amyris Oil is a mixture of chemicals (UVCB), the time window should therefore not be applied to this multi-constituent substance (OECD, 2006)

Conclusions:

Amyris Oil was biodegraded by 62% at day 28 in the Manometric Respirometry Test and should be classified as readily biodegradable.

Executive summary:

The ready biodegradability was determined in the Manometric Respirometry test (OECD TG 301F) performed according to slightly modified OECD, EU and EPA Test Guidelines, and in compliance with GLP. Amyris Oil did not inhibit the intrinsic respiration of the inoculum at the test concentration. Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days. Amyris Oil was biodegraded by 62% at day 28 (63% after 35 days). Amyris Oil is a mixture of chemicals (UVCB), the time window concept should therefore not be applied to this multi-constituent substance (OECD, 2006). Amyris Oil should be classified as readily biodegradable.