Fir, Abies balsamea, 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:

18 May 2018 - 15 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Deviations:

yes

Remarks:

The difference of extremes of replicate values of the removal of the test substance at the plateau (Day 28) was greater than 20 % during the test. No presumed effect.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

yes

Remarks:

The difference of extremes of replicate values of the removal of the test substance at the plateau (Day 28) was greater than 20 % during the test. No presumed effect.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 February 2017

Deviations:

yes

Remarks:

The difference of extremes of replicate values of the removal of the test substance at the plateau (Day 28) was greater than 20 % during the test. No presumed effect.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoid contact with iron.

Study design

Oxygen conditions:

not specified

Inoculum or test system:

sewage, domestic, adapted

Details on inoculum:

The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.
Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

2.3 mg/L

Based on:

test mat.

Remarks:

Based on measured COD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

RECONSTITUTED WATER

1) Stock solutions

In deionised water, analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125 g (VWR PROLABO, Lot# 17B134115, Exp 31 January 2022)
K2HPO4 5.4375 g (VWR PROLABO, Lot# 17K164125, Exp 25 September 2022)
Na2HPO4 x 12H2O 16.795 g (VWR PROLABO, Lot# 17F274113, Exp 08 March 2022)
NH4Cl 0.125 g (SIGMA ALDRICH, Lot# BCBM9152V, Exp 29 February 2020)
Deionised water ad 250 mL
b) Solution: MgSO4 x 7 H2O 5.625 g (VWR PROLABO, Lot# 17A184115, Exp 31 December 2021)
Deionised water ad 250 mL
c) Solution: CaCl2 x 2 H2O 9.10 g (VWR PROLABO, Lot# 16L154110, Exp 31 August 2021)
Deionised water ad 250 mL
d) Solution: FeCl3 x 6 H2O 0.25 g (SIGMA ALDRICH, Lot# BCBJ9853V, Exp 30 September 2018)
Deionised water ad 1000 mL

2) Ratio of ingredients

Adequate amount of the stock solutions a) - d) were combined and filled up with deionised water to the appropriate final volume (ratio of composition referring to 1 mL of the stock solutions a) - d) filled up with deionised water to a final volume of 1000 mL according to the guideline). The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was 8.5 mg/L at about 22 °C.

TEST UNITS

Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.


TEST CONDITIONS

The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 +- 2 °C according to guideline. The test flasks were placed into an incubator and kept at 20.0 - 23.4 °C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 8.5 mg/L at the start of the test.
The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.38.

The test conditions were measured with suitable instruments and documented in the raw data.


PREPARATION OF THE TEST SOLUTIONS


The respective amount of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) was weighed in directly to reach the required test item concentration of 2.3 mg/L.
The chosen test item concentration was based on measured chemical oxygen demand (COD): 2.59 ±0.060 mg test item and on the performed 14-d preliminary test.

The components were applied in the amounts/volumes in the test flasks:

1.) Test Item (flasks 1a and 1b)

Based on the measured chemical oxygen demand (COD) of 2.59 mg O2/mg test item, 7.8 mg of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 2.3 mg/L test item, with a COD of about 5.957 mg O2/L).

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)

Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.168 mg of Sodium benzoate was mixed into 3.38 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).

* The concentration of the stock solution was: 360 mg/L.

3.) Inoculum Control (flasks 3a and 3b)

Only filtered inoculum was added to 3.38 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)

The Test item (7.8 mg) and the reference item stock solution* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 2.3 mg/L test item (COD of 5.957 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).

* The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.


COURSE OF THE TEST

1. Preparation of Test Flasks

Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

2. The Test Bottles

The number of test bottles was the follow:
 10 bottles containing the test item and inoculum
 10 bottles containing the reference item and inoculum (procedure control)
 10 bottles containing only inoculum (inoculum control)
 10 bottles containing the test item, reference item and inoculum (toxicity control)

MEASUREMENTS

1. Measurement of Oxygen

The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

2. Measurement of Total Oxidised N, Nitrite and Nitrate

The concentration of dissolved oxygen resulted a mean of 7.80 mg O2/L after 14 days of incubation in the test item flasks (1a and 1b), which resulted a mean of 3.4 % biodegradation within 14 days of the test item during the performed preliminary experiment, therefore samples for nitrate and nitrite analysis were not taken and measurement of the total oxidised Nitrogen (nitrate and nitrite) concentrations was not performed.

3. Measurement of Temperature

Temperature was measured continuously and registered on weekdays.

Reference substance

Reference substance:

other: Sodium benzoate, C7H5NaO2, Batch # A0371807, Exp: 30 April 2019, ACROS ORGANICS

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Remarks:

Test item initial concentration is 2.3 mg/L

Value:

8.3

Sampling time:

0 d

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

7.65

Sampling time:

7 d

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

7.2

Sampling time:

14 d

% Degradation 4

Parameter:

% degradation (O2 consumption)

Value:

7.1

Sampling time:

21 d

% Degradation 5

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

28 d

Details on results:

DEVIATION FROM THE STUDY PLAN AND TEST GUIDELINE
 
Concerning: Conditions for the validity of the test

According to the Study Plan: The difference of extremes of replicate values of the removal of the test substance at the plateau, (at the end of the test or at the end of the 10 or 14-d window) will be less than 20 %.

Deviation: The difference of extremes of replicate values of the removal of the test substance at the plateau (Day 28) was greater than 20 % during the test.

Reason for this change: Deviation between replicates is relatively large because low degradation values cause large percentage differences.

Presumed Effect on the Study: None.Taking into account the very low biodegradation rate calculated, and that the test item was not considered to have significant inhibitory effects. Therefore this deviation is negligible and it has no impact on the results of the study, consequentlythe study is considered to be valid.

BOD5 / COD results

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 66.7 % after 14 days, and to a mean of 74.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

Any other information on results incl. tables

Table: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Flask	Percent of biodegradation after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item		1a	-1.7	6.7	5.9	4.2
	2.3	1b	0.0	6.7	5.9	7.6
		mean	-0.8	6.7	5.9	5.9
Reference item		2a	60.0	66.7	74.2	74.2
	3.6	2b	60.0	66.7	72.5	74.2
		mean	60.0	66.7	73.3	74.2
Toxicity control	Test item: 2.3 Reference item: 3.6	4a	29.5	34.2	36.2	37.8
		4b	29.5	34.2	37.0	39.4
		mean	29.5	34.2	36.6	38.6

Biodegradation % = [BOD of T or R / (COD of T or ThOD of R)]*100

where:

T         = test item

R         = reference item

COD of test item        = 2.59 ±0.060 mg O₂/mg test item

ThOD of reference item = 1.67 mg O₂/mg reference item

The biodegradation in the toxicity control was calculated according to the following formula: [BOD of T and R / (COD of T + ThOD of R)*0.5]*100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item Fir Needle Oil Canadian, is considered not readily biodegradable.

Executive summary:

The test item Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 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.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) reached a mean of 5.9 % after 28 days based on the COD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD/COD. Therefore the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 66.7 % after 14 days, and to a mean of 74.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 34.2 % biodegradation was noted within 14 days and 38.6 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.3 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.