Pine, Pinus sylvestris, ext.

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD Guideline 301 C but with deviations: no detail on material and methods

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

yes

Remarks:

no detail on material and methods

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Concentration of sludge: 30 ppm

Duration of test (contact time):

14 d

Initial conc.:

100 other: ppm

Based on:

test mat.

Details on study design:

No data

Preliminary study:

No data

Test performance:

No data

Parameter:

% degradation (O2 consumption)

Value:

>= 41 - <= 98

Sampling time:

14 d

Details on results:

- Indirect analysis (BOD): 41, 81 and 98%
- Direct analysis (GC): 100%
- Direct analysis (TOC): 50, 73 and 81%

Results with reference substance:

No data

None

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, Limonene was readily biodegradable.

Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, Limonene was tested at concentrations of 100 ppm and the inoculum was activated sludge (30 ppm). The degradation of the test material was assessed by the determination of the oxygen consumption.

At 100 ppm test concentration, 41-98% degradation by biochemical oxygen demand was reached in 14 days.

 

Under the test conditions, Limonene was readily biodegradable.

 

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD Guideline 301 C but with deviations: no detail on material and methods

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

yes

Remarks:

no detail on material and methods

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Concentration of sludge: 30 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Details on study design:

No data

Preliminary study:

No data

Test performance:

No data

Parameter:

% degradation (O2 consumption)

Value:

>= 90 - <= 95

Sampling time:

28 d

Details on results:

- Indirect analysis (BOD): 91, 90 and 95%
- Direct analysis (GC): 100, 100 and 100%

Results with reference substance:

No data

None

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, alpha-pinene was readily biodegradable.

Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, alpha-pinene was tested at concentrations of 100 mg/L and the inoculum was activated sludge (30 mg/L). The degradation of the test material was assessed by the determination of the oxygen consumption.

At 100 mg/L test concentration, 90 -95% degradation (biochemical oxygen demand) was reached in 28 days.

 

Under the test conditions, alpha-pinene was readily biodegradable.

 

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 09 to July 08, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD guideline 301 D with only minor deviations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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 (omission does not result in nitrogen limitation); activated sludge instead of an effluent/extract/mixture was used as inoculum

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of secondary activated sludge: Activated sludge plant treating predominantly domestic waste water (Nieuwgraaf, Duiven, The Netherlands)
- Preconditioning: 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week and then diluted in the BOD bottles
- Concentration of sludge: 400 mg DW/L

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Nutrient medium contained per liter of deionised water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCI2, 0.25 mg FeCI3.6H2O
- Source/preparation of dilution water: Deionised water containing no more than 0.01 mg/L Cu was prepared in a water purification system.
- Test temperature: 22-24 °C
- pH (at start of test): 7.0; pH (at end of test): 6.8 (control, control with silica gel and test)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel dosed with test substance, and 6 bottles containing sodium acetate and inoculum
- Test performed in closed vessels due to significant volatility of test substance: Yes

MEASURING EQUIPMENTS:
- Dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands)
- pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Germany).
- Temperature was measured and recorded with a sensor connected to a data logger.

SAMPLING
- Sampling frequency: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Days 0, 7, 14, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; containing inoculum only
- Procedure control: Yes; containing reference substance (sodium acetate) with inoculated medium
- Toxicity control: No; test material was considered to be non-toxic to micro-organisms as inhibition of the endogenous respiration of the inoculum was not detected during the biodegradation test
- Other: Containing inoculum and silica gel

Reference substance:

acetic acid, sodium salt

Remarks:

6.7 mg/L; source: Acros organics, Belgium; purity: > 99%

Preliminary study:

No data

Test performance:

The validity of the test is demonstrated by an endogenous respiration of 1.0 mg/L at day 28. Sodium acetate was degraded 83% of its ThOD after 28 days. The validity of the test is also shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Details on results:

Initial test material concentration: 2 mg/L
- Theoretical oxygen demand (ThOD) = 3.3 mg/mg
- % biodegradation on Day 3: > 10%
- % biodegradation on Day 13: > 60%
- % biodegradation on Day 28 = 76%

Results with reference substance:

- Theoretical oxygen demand (ThOD) = 0.8 mg/mg
- % biodegradation on Day 1: > 10%
- % biodegradation on Day 6: > 60%
- % biodegradation on Day 14 = 83%

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles

 

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.6	8.6	8.6	8.6
	8.6	8.6	8.6	8.6
Mean (M)	8.6	8.6	8.6	8.6
7	7.8	5.7	8	3.9
	8	5.7	7.9	3.9
Mean (M)	7.9	5.7	8	3.9
14	7.8	3.3	7.7	3.4
	7.6	3.5	7.8	3.2
Mean (M)	7.7	3.4	7.8	3.3
21	7.7	3	7.8
	7.6	2.8	7.6
Mean (M)	7.7	2.9	7.7
28	7.6	2.6	7.5
	7.6	2.6	7.6
Mean (M)	7.6	2.6	7.6

O_cs: Mineral nutrient solution without test material but with inoculum and silica gel

O_t: Mineral nutrient solution with test material (2.0 mg/L), silica gel, and inoculum

O_c: Mineral nutrient solution with only inoculum

O_a: Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum

 

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, β-pinene (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
7	2.2	4.1	33	76
14	4.3	4.5	65	83
21	4.8		74
28	5		76

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration at Day 28 was 1.0 mg/L; differences of the replicate values at Day 28 were < 20%; degradation in reference material was 83% at Day 14; O2 concentration during the test was > 0.5 mg/L

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, β-pinene was readily biodegradable.

Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 D and GLP, β-pinene was tested at concentrations of 2 mg/L and the inoculum was activated sewage sludge, domestic. The degradation of the test material was assessed by the determination of the oxygen consumption. The test treatments, inoculum blank, and reference (sodium acetate) were measured in duplicates.

 

At 2 mg/L test concentration, greater than 10% biodegradation of the test substance was reached on Day 3 and greater than 60% biodegradation was reached on Day 13. Hence, the test material met the 14 day window requirement for ready biodegradability. On Day 28, the biodegradation was 76%.

 

The reference material, sodium acetate, reached greater than 60% biodegradation on Day 6. β-pinene was considered to be non-toxic to micro-organisms as inhibition of the endogenous respiration of the inoculum was not detected during the test. Hence, the study met the validity criteria for reference material and toxicity control. The endogenous respiration at Day 28 was 1.0 mg/L and oxygen concentration during the test was greater than 0.5 mg/L.

 

Under the test conditions, β-pinene was readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2013-06-17 to 2013-07-31

Reliability:

1 (reliable without restriction)

Justification for type of information:

Experimental data on substance with similar composition than registered substance.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Aerobic activated sludge (microorganisms from a domestic wastewater treatment plant) was supplied by the sewage works of Rossdorf, Germany.
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap 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. This suspension was aerated overnight and was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

78 mg/L

Based on:

test mat.

Initial conc.:

236.7 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test flasks were incubated at 22 ± 1 °C. The The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days. 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 O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

Test conditions: climatised room 22 +-1°C
Light: Darkness
pH-value of thest solutions: 7.6 (measured at the start of the test ; 7.6 - 7.8 (measured at the end of the test)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

no data

Test performance:

no data

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

28 d

Remarks on result:

other: failing 10-day window

Details on results:

no data

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 91% after 28 days of incubation.

no data

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Pine needle oil undergoes 70% biodegradation after 28 days in the test conditions. Biodegradation starts on day 3 and reached 70% at the end of the 28-day period (10-day window failed). The curves obtained with the reference substance alone and with Pine needle oil + reference substance show no toxic effect of Pine needle oil on the micro-organisms at the test concentration.

Executive summary:

Pine needle oil undergoes 70% biodegradation after 28 days in the test conditions in a 301F study. Biodegradation starts on day 3 and reached 70% at the end of the 28-day period.

The degradation rate of Pine needle oil did not reach 60% within the 10-day window, but after 28 days. Therefore, Pine needle oil is considered to be readily biodegradable, but failing 10-day window.

Due to the fact that the substance is identified as an UVCB or as a complex, 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.

The curves obtained with the reference substance alone and with Pine needle oil + reference substance show no toxic effect of Pine needle oil on the micro-organisms at the test concentration. Therefore under the test condition Pine needle oil is considered as ready biodegradable.

Reference 5

Endpoint:

biodegradation in water: screening test, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2014-08-19

Reliability:

2 (reliable with restrictions)

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Qualifier:

no guideline required

Principles of method if other than guideline:

QSAR, Biowin v4.10. More details are given in QMRF/QPRF attached to the dossier.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Details on inoculum:

not applicable

Details on study design:

not applicable

Preliminary study:

not applicable

Test performance:

not applicable

Remarks on result:

other: Biowin 1 = Does Not Biodegrade Fast Biowin 2 = Does Not Biodegrade Fast Biowin 3 = Weeks – Months Biowin 4 = Days - Weeks Biowin 5 = Readily Degradable Biowin 6 = readily Degradable Biowin 7 = Does Not Biodegrade Fast

Details on results:

Biowin 1 = Does Not Biodegrade Fast
Biowin 2 = Does Not Biodegrade Fast
Biowin 3 = Weeks – Months
Biowin 4 = Days - Weeks
Biowin 5 = Readily Degradable
Biowin 6 = readily Degradable
Biowin 7 = Does Not Biodegrade Fast

Results with reference substance:

not applicable

Validity criteria fulfilled:

not applicable

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Biowin 1 = Does Not Biodegrade Fast
Biowin 2 = Does Not Biodegrade Fast
Biowin 3 = Weeks – Months
Biowin 4 = Days - Weeks
Biowin 5 = Readily Degradable
Biowin 6 = readily Degradable
Biowin 7 = Does Not Biodegrade Fast

Executive summary:

Delta-3 -carene ready biodegradation property was investigated using QSAR approach with Biowin v4.10 plug-in from EPISUITE v4.1 from USEPA.

Results are as follow:

Biowin 1 = Does Not Biodegrade Fast

Biowin 2 = Does Not Biodegrade Fast

Biowin 3 = Weeks – Months

Biowin 4 = Days - Weeks

Biowin 5 = Readily Degradable

Biowin 6 = readily Degradable

Biowin 7 = Does Not Biodegrade Fast

Therefore, it is expected that Delta-3 -carene is not readily biodegradable.

Description of key information

Based on the results from ready biodegradability tests and prediction of delta-3-carene showing that all major constituents are readily biodegradable, it can be concluded that Pine scotch oil is expected to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

 All major constituents of Pine scotch oil are considered readily biodegradable, except delta-3-carene (see table below). However, it has been shown, with a similar (in composition) substance (Pine needle oil) that contains 13.30 % of delta-3-carene, that once in mixture delta-3-carene does not inhibit the ready biodegradability property of the substance.

Constituents	Typical composition	Biodegradation results	Reference in IUCLID 6
(d,l)-limonene	6.50 %	Readily biodegradable	(d,l)-limonene, 138-86-3, Biodegradation in water: screening tests, MITI, 1980, RS, W
alpha-pinene	55 %	Readily biodegradable	alpha-pinene, 80-56-8, Biodegradation in water: screening tests, MITI, 2007, RS, W
beta-pinene	24 %	Readily biodegradable	beta-pinene, 127-91-3, Biodegradation in water: screening tests, van Ginkel, 2010, RS, W
Delta-3-carene	10 %	Not Readily biodegradable	Delta-3-carene, 13466-78-9, Biodegradation in water, screening test, Biowin v4.10, RS, W
Pine needle oil	N/A	Readily biodegradable	Biodegradation in water, Pine needle oil, 2013, RS, W

 

 

Therefore, based on a Weight of Evidence approach, it can be possible to consider that Pine scotch oil is readily biodegradable.