Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

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:

From June 22, 2020 to September 09, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Copy of GLP certificate included with date of inspection: 27.06, 21-23.10.2019

Specific details on test material used for the study:

Theoretical Oxygen Demand:ThODNH4: 2.723 mg oxygen per mg test item

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Species/Origin:Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.

Sludge preparation: The aerobic activated sludge used for this study was deposited for 10 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnightat room temperature. Thissuspension was used for the experiment.

Duration of test (contact time):

60 d

Initial conc.:

49.3 mg/L

Based on:

test mat.

Initial conc.:

48.4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Units
- Type and Size:Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Apparatus:BSB-Sensomat-System©
- Principle:The test flasks were prepared according paragraph 6.6 and were incubated at 22°C ± 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 60 days using the BSB-Sensomat-System©. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2consumed by the activated sludge was calculated from thedecrease of pressure in the reaction vessel.
- Identification:Each test unit was uniquely identified with the study number, treatment and replicate number.

Test Conditions
- Surrounding Type:Climatised chamber
- Temperature:22°C ± 1°C
- Light Conditions:Darkness
- pH-Value of Test Solutions:7.7 (measured at the start of the test), adjusted to 7.4 and 7.5 with HCl) 7.5 to 7.9 (measured at the end of the test)
- Recording:Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (≤2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and are not reported.)

Test Water
- Reconstituted Test Water:Analytical grade salts were added to ultrapure water to prepare the following stock solutions:
a)8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; the pH-value was 7.4.
b)11.25 g MgSO4x 7 H2O filled up with pure water to 500 mL volume
c)18.2 g CaCl2x2 H2O filled up with purewater to 500 mL volume
d)0.125 g FeCl3x 6 H2O filled up with purewater to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solutiond), one drop of concentrated HCl per litre was added before storage. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with ultrapure water.
The pH was 7.6 and therefore no adjustment was necessary.

Course of the Test
- Preparation of Test Flasks:The amounts of test item and reference item were directly weighed into the test flasks. For a better distribution of the test item, silicone oil AR 20 at a concentration of about 1% of the final flask volume was added to all flasks in order to improve the bioavailability of the mixture constituents.
- Incubation:The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Test Duration:60 days

Test Parameters
- Measurement of Oxygen:The change of pressure in the test flasks was measured by means of a manometric method (BSB-Sensomat-System©).
- Temperature:The temperature was recorded by means of the automated software AMR Wincontrol©.
- pH-Value:pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.


Result Evaluation
Definitions:
- ThODNH4: Theoretical Oxygen DemandThODNH4 is the total amount of oxygen required to oxidise a chemical completely. It is calculated from the molecular formula (assuming no nitrification occurs) and expressed as mg oxygen required per mg test item.
- BOD:Biochemical Oxygen DemandBOD is the amount of oxygen consumed by microorganisms when metabolising a test item; also expressed as mg oxygen uptake per mg test item/L.
- Conversion Factor:Conversion Factor is necessary for the calculation of the BOD and depends on the final volume in the test flasks and is predetermined by the BSB-Sensomat-System© supplier.At a test volume of 244 ml the conversion factor is 5.
- 10-day window:The 10 day window is the 10 day period immediately following the attainment of 10 % biodegradation.
If the substance is identified as an UVCB or as a complex, multi-constituent substance with structurally similar constituents, the 10-day window condition may be waived and the pass level applied at 28 days according to section 4.1.2.9.5 of CLP COMMISSION REGULATION (EU) No 286/2011 of 10 March 2011.

BOD [mg/L] =Measured data * conversion factor
BODControl adjusted = BOD of test item (or reference item) – mean BOD of inoculum controls
ThOD [mg O2/L] = Test item or reference item [mg] * ThOD [mg O2/mg test item or reference item] / 0.244L
The percentage biodegradation of the test item and of the reference item sodium benzoate is calculated as:
% degradation =BODControl adjusted [mg/L] * 100/ThOD [mg O2/L]
For the toxicity control, the ThOD is the sum of the ThOD of the test item and the ThOD of the reference item weighed in the toxicity control flask.

Validity Criteria of the Study
- Inoculum Control:The oxygen demand of the inoculum control (medium and inoculum) was 27.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
- pH-Value:The pH-value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
- Reference Item:The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.The reference item sodium benzoate was degraded to more than 60% after 4 days and to80% after 14 days of incubation.
- Test Item:The difference of duplicate values for the degradation of the test item at the plateauafter 45 days andat the end of the test was less than 20%. The difference of duplicate values at days 45 and 60 was 1% and 0%, respectively. The validity criterion was fulfilled.
- Toxicity Control:If in a toxicity test, containing both the test item and a reference itemless than 25% biodegradation (based on ThODNH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 41% at day 14; the test item was not inhibitory.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

57

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

60 d

Details on results:

The mean biodegradation of ORANGE ESS TERPENLESS TYPE BRAZIL 015231 at day 28 was 57% (ThODNH4), therefore the study was prolonged till day 60. A mean degradation of more than 60% was reached at day 33. The mean biodegradation at test end after 60 days was 77% (ThODNH4).

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 80% after 14 days and to 84% after 28 days of incubation.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Biodegradation in the Toxicity Control

- Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item sodium benzoate, 41% (ThOD Nh4) biodegradation was noted within 14 days, 53% biodegradation after 28 days (ThODNH4 ) and 57% after 60 days of incubation (ThODNH4).

- Conclusion: According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25% within 14 days.

Abiotic Control

- Oxygen Demand: The oxygen demand in the abiotic cotrol was 0 mg/L during the test duration.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The degradation rate of ORANGE ESS TERPENLESS TYPE BRAZIL 015231 did not reach 60% after 28 days, but after 60 days of incubation. Therefore, ORANGE ESS TERPENLESS TYPE BRAZIL 015231 is considered to be not readily biodegradable based on ThODNH4 after 28 days but reached a degradation rate of 77% (ThODNH4) after 60 days.

Executive summary:

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 F: "Ready biodegradability: Manometric Respirometry Test" over a period of 60 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 60 days of incubation. The test item loading rate was 102.7 mg/L (corresponding to an oxygen demand of about 279.6 mg/L (ThODNH4).

The mean biodegradation of the test item at day 28 was 57% (ThODNH4), therefore the study was prolonged till day 60. A mean degradation of more than 60% was reached at day 33. The mean biodegradation at test end after 60 days was 77% (ThODNH4). Therefore, test item is considered to be not readily biodegradable based on ThOD NH4 after 28 days but reached degradation rate of 77% (ThODNH4) after 60 days.

Description of key information

OECD Guideline 301F, GLP, key study, validity 1:

57% after 28 days, 77% after 60 days.

The substance is not readily biodegradable but significant mineralisation occurred thus demonstating complete primary biodegradation, and based on the enhanced screening test, extended up to 60 days, the substance is not persistent in the environment.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

One key valid experimental study is available to assess the biodegradation in water of the test substance:

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 F: "Ready biodegradability: Manometric Respirometry Test" over a period of 60 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 60 days of incubation. The test item loading rate was 102.7 mg/L (corresponding to an oxygen demand of about 279.6 mg/L (ThODNH4).

The mean biodegradation of the test item at day 28 was 57% (ThODNH4), therefore the study was prolonged till day 60. A mean degradation of more than 60% was reached at day 33. The mean biodegradation at test end after 60 days was 77% (ThODNH4). Therefore, test item is considered to be not readily biodegradable based on ThOD NH4 after 28 days but reached degradation rate of 77% (ThODNH4) after 60 days.