Cypress, Cupressus funebris, 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:

13-09-2016 to 12-10-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification and river water instead of an effluent/extract/mixture was used as inoculum.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cypress, Cupressus funebris, ext.

EC Number:

285-360-9

EC Name:

Cypress, Cupressus funebris, ext.

Cas Number:

85085-29-6

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood obtained from the wood of Cupressus funebris (Cupressaceae) by steam distillation

Test material form:

other: Pale yellow to yellow liquid

Remarks:

could crystallize

Details on test material:

- Name of test material (as cited in study report): Cedarwood Oil China
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information

Specific details on test material used for the study:

Identification: (Trade Name) Cedarwood oil Chinese
Purity: 100% UVCB
Batch/Lot number: 1002515317
Expiry date: 31-08-2017
Molecular formulae: C15H24, C15H22, C15H26O, based on known constituents(~85%)
Solubility in water: insoluble in water
Stability: not relevant in biodegradation tests
Storage at ambient temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum/activated sludge: River water was sampled from the Rhine near Heveadorp, The Netherlands (07-09-2016). 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.

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 test substance concentration

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

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 no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.

Nutrients, stocks and administration:
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the 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 top and the content was mixed vigorously. 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 nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L

Test procedure:
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 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 completely 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 all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Test conditions:
The pH of the media was 8.0 at the start of the test. The pH of the media at day 28 was 7.9 (test and control with silica gel) and 8.0 (control). Temperatures were within the prescribed temperature range of 22 to 24°C.

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

purity > 99% Batch No. BCBP8197V

Results and discussion

Test performance:

The validity of the test is demonstrated by an endogenous respiration of 1.4 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 85. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

72

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

0 d

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

7 d

% Degradation 4

Parameter:

% degradation (O2 consumption)

Value:

39

Sampling time:

14 d

% Degradation 5

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

21 d

Details on results:

Cedarwood Chinese oil was biodegraded by 72% at day 28 in the Closed Bottle test. The time-day window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Cedarwood Chinese oil is a mixture of chemicals (UVCB). The biodegradation kinetics (lag period, growth rate, and yield) of the individual chemicals in a mixture are not necessarily same. The biodegradation of a fragrance substance consisting of many constituents is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the multi-constituent suggests that the test substance is not readily biodegradable. The time window that can be applied to Closed Bottle tests is therefore not considered applicable to this multi-constituent substance (OECD, 2006), even though it is met. Over 60% biodegradation was achieved in a period of approximately 14 days immediately following the attainment of 10% biodegradation at day 5, therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds. Cedarwood Chinese oil should be classified as readily biodegradable.

reference:
*OECD (2006) Revised introduction to the OECD guidelines for testing of chemicals, section 3, Part 1: Principles and strategies related to the testing of degradation of organic chemicals, Paris Cedex, France.

BOD5 / COD results

Results with reference substance:

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of Cedarwood oil Chinese to microorganisms degrading acetate is not relevant. 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.

Any other information on results incl. tables

Mean (n=2) dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.7	8.7	8.7	8.7
7	7.9	7.0	7.8	3.9
14	7.6	5.1	7.4	2.8
21	7.4	3.0	7.4
28	7.3	2.7	7.3

Ocs: River water with nutrients and silica gel.

Ot: River water with nutrients, test material (2.0 mg/L) and silica gel.

Oc: River water with nutrients.

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

 

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, Cedarwood Chinese oil (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.9	3.9	14	72
14	2.5	4.6	39	85
21	4.4		69
28	4.6		72

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See "test performance" for details

Interpretation of results:

readily biodegradable

Remarks:

Cederwood oil Chinese is a mixture of chemicals (UVCB), the time window applicable for the closed bottle test should therefore not be applied to this multi-constituent substance (OECD, 2006).

Conclusions:

Cedarwood oil Chinese should be classified as readily biodegradable, since 72% degradation was observed at 28 d.

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. Cedarwood oil Chinese 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. Cedarwood oil Chinese was biodegraded by 72% at day 28 in the OECD 301 Closed Bottle test. The substance should therefore be classified as readily biodegradable