Cedrus atlantica, ext.

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:

03/08/2012-09/11/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, Part 1, 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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, Switzerland)
- Storage length: usage on the same day as collection of the inoculum
- Pretreatment: sludge was collected in the morning and washed three times in the mineral medium (centrifuging at 1000g for 10 min, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
- Concentration of the inoculum in the test medium was 30 mg/l (dry weight)

Duration of test (contact time):

54 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of mineral medium: The mineral medium was prepared by mixing 50ml of solution A and 2000ml deionised water, adding 5ml of each of the solutions B,C and D and making up to 5 litres with deionised water.

Solution A:
KH2PO4 8.5g
K2HPO4 21.75g
Na2HPO4*2H2O 33.4g
NH4Cl 0.5g
dissolved in water and made up to 1 litre

Solution B:
CaCl2 27.5g
dissolved in water and made up to 1 litre

Solution C:
MgSO4*7H2O 22.5g
dissolved in water and made up to 1 litre

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

- Test temperature: 22.3-23.5°C ± 1°C
- pH: 7.4 ± 0.2
- pH adjusted: yes, if necessary with phosphoric acid or potassium hydroxide
- Aeration of inoculum: yes, until being used in the test
- Suspended solids concentration: 3,56 g/l dw, diluted to 1,53 g/l
- Continuous darkness: yes/no

TEST SYSTEM
- Number of culture flasks/concentration: 6 flasks with 30mg test substance/l
- Test substance was directly added to the test flasks of the Oxitop. No efforts were carried out to increase the bioavailability.
- Measuring equipment: Oxitop Control System as Respirometer, made by WTW, Weilheim, Germany
- Test performed in closed vessels: yes, flasks were closed tightly with measuring heads.
- Test performed in open system: no
- Details of trap for CO2: evolved carbon dioxide is absorbed in sodium hydroxide pellets
- Other: the consumption of oxygen is determined by measuring the pressure drop in the respirometer flask; The Biological Oxygen Demand (BOD) is expressed as a percentage of ThOD (Theoretical Oxygen Demand).

SAMPLING
- Sampling frequency: Every day the oxygen consumption of each flask is recorded and the correct temperature and stirring checked.

CONTROL AND BLANK SYSTEM
- Reference substance as control: yes (100mg/l)
- Toxicity control: no


STATISTICAL METHODS:
Oxygen uptakes, as read on the Oxitop controller, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.

Reference substance:

benzoic acid, sodium salt

Remarks:

Fluka, Buhs, Switzerland, Art. No. 71300; Purity min. 99%

Parameter:

% degradation (O2 consumption)

Value:

9

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

24

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

47

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

42 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

54 d

Details on results:

The O2 uptake of test substance was measured on day 7, 14, 21, 28, 35, 42 and 54 with % degradation results of 9, 24, 36, 47, 54, 61 and 70, respectively.

Results with reference substance:

The O2 uptake of the reference substance was measured on day 5, 7, 14, 21 and 28 with % biodegradability results of 72, 78, 88, 92 and 94 respectively.

O2 uptake (mg/l, adjusted to normal concentrations) of Test Substance:

Theoretical Oxygen Demand (ThOD): 3.21 mg O2/mg

Days:  7  14  21  28  35  42  54  O2 Uptake of sludge (inoculum blank)  1  B1  21.5  28.3  32.3  35.0  37.7  41.7  44.4    2  B2  22.9  28.3  32.3  35.0  36.3  39.0  41.7    mean  B  22.2  28.3  32.3  35.0  37.0  40.4  43.1  O2 Uptake of Test Subst. + sludge  9  C1  29.6  49.8  64.6  76.6  84.7  94.1  107.9    10  C2  32.3  53.8  70.0  83.4  92.8  103.9  113.9  O2 Uptake of Test Substance    C1 -B  7.4  21.5  32.3  41.6  47.7  53.8  64.9      C2 -B  10.1  25.5  37.7  48.4  55.8  63.6  70.9  % Biodegradation of test substance    D1  8  22  33  43  50  56  67      D2  10  26  39  50  58  66  74    mean  D  9  24  36  47  54  61  70

 O2 uptake (mg/l, adjusted to nominal concentrations) of Reference Substance:

Theoretical Oxygen Demand (ThOD): 1.67 mg O2/mg

Days:  5 7  14  21  28  O2 uptake of sludge (inoculum blank)  1  B1  18.8  21.5  28.3  32.3  35.0    2  B2  20.2  22.9  28.3  32.3  35.0    mean  B  19.5  22.2  28.3  32.3  35.0  O2 uptake of Refer. subst. + sludge  17  A1  131.1  146.2  167.4  178.4 183.4     18  A2  146.2  157.3  183.5  193.6  198.6  O2 uptake of Refer. substance    A1 -B  111.6  124.0  139.1  146.1  148.4      A2 -B  126.7  135.1  155.3  161.3  163.6  % biodegradation of reference substance    D1  67  74  83  88  89      D2  76  81  93  97  98    mean  D  72  78  88  92  94

Validity criteria fulfilled:

yes

Remarks:

Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days, the activity of the inoculum was verified. The repeatability criterion (not more than 20% differ. between replicates) is fulfilled for the flasks containing test substance.

Interpretation of results:

other: inherently and ultimately biodegradable

Conclusions:

In a screening test for ready biodegradation, Cedarwood Oil Atlas Orpur reached 45% of the theoretical oxygen consumption after 28 days. After 42 days it has passed 60% and 70% after 54 days. Thus the test substance is not readily biodegradable under the stringent conditions in this test, but it is considered to be inherently and ultimately biodegradable.

Executive summary:

The Ready Biodegradability of Cedarwood Oil Atlas Orpur was determined by the Manometric Respirometry Test (OECD TG 301F, 1992). The test substance did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration. Cedarwood Oil Atlas Orpur showed 47% oxygen consumption after 28 days under the stringent conditions of this test. Prolongation of the test resulted in 70% oxygen consumption after 54 days. It is concluded that Cedarwood Oil Atlas Orpur is not readily biodegradable, but it is inherently and ultimately biodegradable.

The reviewer remarks that the degradation curve has an atypical almost linear shape with a lag phase. Cedarwood Atlas oil is actually a mixture of constituents with differing solubility in water. This means that a proportion of the substance will be in the undissolved phase and hence, not directly available to the degrading organisms. Transport will be limited by diffusion limitation due to the poor water solubility of the constituents. No efforts were made to increase the bioavailability of the oil to the degrading organisms. This results in an underestimate of the true potential to biodegrade in the environment, as well as in an underestimate of the rate of degradation. Moreover, Cedarwood Atlas oil is a complex mixture which results in a stepwise, sequential adaptation of the microorganisms to utilise the individual constituents. For these reasons, typical logarithmic growth phase biodegradation kinetics may not be observed, so even if individual constituents are readily biodegraded, the NCS may not achieve the 10 -d window (see ECHA Guidance R.7b, Appendix 7.9 -4 on Testing of Mixtures). Thus, considering that the oxygen consumption of 47% after 28 days and 61% in 42 days is probably an underestimation of the true degradation potential and degradation rate, the substance is expected to degrade rapidly in the environment.

Description of key information

Cedarwood Atlas Oil was tested in an enhanced screening test for ready biodegradation. Under these stringent test conditions, the NCS was not readily biodegradable but it is found to be inherently and ultimately biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

The Ready Biodegradability of Cedarwood Oil Atlas Orpur was determined by the Manometric Respirometry Test (OECD TG 301F, 1992, Key study, Rel. 1). The test substance did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration. Cedarwood Oil Atlas Orpur showed 47% oxygen consumption after 28 days under the stringent conditions of this test. Prolongation of the test resulted in 70% oxygen consumption after 54 days. It is concluded that Cedarwood Oil Atlas Orpur is not readily biodegradable, but it is inherently and ultimately biodegradable.

The degradation curve has an atypical almost linear shape with a lag phase. Cedarwood Atlas oil is actually a mixture of constituents with differing solubility in water. This means that a proportion of the substance will be in the undissolved phase and hence, not directly available to the degrading organisms. Transport will be limited by diffusion limitation due to the poor water solubility of the constituents. No efforts were made to increase the bioavailability of the oil to the degrading organisms. This results in an underestimate of the true potential to biodegrade in the environment, as well as in an underestimate of the rate of degradation. Moreover, Cedarwood Atlas oil is a complex mixture which results in a stepwise, sequential adaptation of the microorganisms to utilise the individual constituents. For these reasons, typical logarithmic growth phase biodegradation kinetics may not be observed, so even if individual constituents are readily biodegraded, the NCS may not achieve the 10-d window (see ECHA Guidance R.7b, Appendix 7.9 -4 on Testing of Mixtures). Thus, considering that the oxygen consumption of 47% after 28 days and 61% in 42 days is probably an underestimation of the true degradation potential and degradation rate, the substance is expected to degrade rapidly in the environment.

The biodegradation of the individual constituents was assessed as well (constituent approach).

Cedarwood Atlas oil from the plant Cedrus Atlantica is a Natural Complex Substance (NCS). With 95.2% of the constituents of the oil known, this NCS can be considered as a well defined NCS. The majority of the constituents of Cedarwood Atlas oil belongs to the group of sesquiterpenes.

For a small fraction typically comprising 4% of the composition, the available experimental data indicate that the constituents are readily biodegradable. For the majority of the constituents, typically comprising 83.2% of the composition, the results obtained in tests on the ready biodegradability show that the constituents are not meeting the strict criteria for ready biodegradation. For these constituents, the biodegradation curves had not reached a plateau level at the end of the 60d-test, showing that biodegradation was still progressing. This is probably related to the poor solubility and high lipophility of the constituents, leading to limited mass transfer and thus a low bioavailability in the test systems. It is concluded that all of the tested sesquiterpenes achieved significant ultimate biodegradation in a prolonged OECD 301F test. This evidence is used to assess the fate of other sesquiterpenes (typically 6.4%) with the same carbon skeletons that are likely to follow similar biodegradation pathways. Thus 90% of the constituent composition is considered as Inherently, ultimately biodegradable; in addition to the 4% which is readily biodegradable. For 1.6% of the constituent composition the biodegradability could not be assessed.

The conclusion of the constituent approach is that the NCS Cedarwood Atlas oil is considered to be inherently and ultimately biodegradable.

The constituent approach leads to the same conclusion as the actual test on the NCS itself illustrating that the constituent approach is a valid one.