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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
See attached the QMRF and QPRF for the QSAR model.

Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Howard, P.H., Boethling, R.S., Stiteler, W.M., Meylan, W.M., Hueber, A.E., Beauman, J.A., Larosche, M.E. 1992. Predictive model for aerobic biodegradability developed from a file of evaluated biodegradation data. Environ. Toxicol. Chem. 11:593-603.
Specific details on test material used for the study:
SMILES: C(C(CC1C2)C2)(C1(C)C)=C
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

BIOWIN1 (Linear Model): 0.4988

BIOWIN2 (Non-Linear Model): 0.3432

Expert Survey Biodegradation Results:

BIOWIN3 (Ultimate Survey Model): 2.6860 (weeks-months)

BIOWIN4 (Primary Survey Model): 3.4977 (days-weeks)

MITI Biodegradation Probability:

BIOWIN5 (MITI Linear Model): 0.4717

BIOWIN6 (MITI Non-Linear Model): 0.3865

Anaerobic Biodegradation Probability:

BIOWIN7 (Anaerobic Linear Model): -0.0855

Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
The substance L-camphene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)
Executive summary:

The substance L-camphene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: Test material was evaluated for degradation by cultures derived from coniferous forest soil, diluted and used directly without any prior enrichment.
- Short description of test conditions: see below
- Parameters analysed / observed: see below
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
Details on inoculum:
- Source of inoculum: The primary inocula for this study were prepared from extracts of soil samples collected from a coniferous forest (soil A).
- Preparation of inoculum for exposure: Soil extracts were prepared by passing soil/water mixtures through a 500-µm sieve, followed by 2 h settling. The resulting supernatants were used as the inoculum.
Initial conc.:
>= 0.5 - <= 3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
other: biomass concentration
Details on study design:
First biodegradation experiment:
Glass flask (2 l) equipped with two glass/Teflon valves and a septum-sealed port was used.
The reactor was flushed with pure oxygen and then 1.4 l oxygen saturated minimal medium was added. Test substance was tested at concentrations in the range 0.5-3 mg/l. After the addition of the undiluted monoterpene, the reactor was then crimp-sealed with Teflon-lined septa. After 24 h equilibration, soil A extract was added to the reactor at 1% (v/v) through the bottom glass/Teflon valve. A sodium-azide-amended control was also set up.
Incubation took place in the dark at 23ºC with continuous mixing using magnetic stirrers (at approx. 300 rpm). At regular intervals, duplicate gas and liquid samples were removed and analyzed for test substance and CO2.

Second biodegradation experiment:
A second experiment was performed using replicate, 18x150-mm (26 ml volume) serum tubes (Bellco Glass, Vineland, N.J.). The tubes were flushed with pure oxygen and crimp sealed with Teflon-lined septa.
Inoculum drawn from the reactor used in the previous experiment was directly injected into replicate serum tubes from a microsyringe and then quickly crimp-sealed. The initial terpene concentration in the replicate tubes was uniform.
Azide amended controls were also prepared and incubated following the procedures used for the live cultures.
Headspace gas analysis of serum tubes showed an oxygen content of up to 90%, which was suficient for the complete mineralization of the terpene at the levels tested. The serum tubes were continuously rotated (at 1 rpm) and incubated in the dark at 23ºC. At diferent intervals analyses were performed by sacrificing duplicate serum tubes.
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.029
Sampling time:
200 h
Remarks on result:
other: Experiment 1 (CSR1)
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.63
Sampling time:
98 h
Remarks on result:
other: Experiment 2 (serum tubes)
Key result
Parameter:
other: Normalized degradation rate (h-1)
Value:
0.042
Sampling time:
98 h
Remarks on result:
other: Experiment 2 (serum tubes)
Details on results:
The detection of CO2, the increase in biomass concentration and lack of any substantial change in the concentration of terpene in the azide-amended control reactor demonstrated that biodegradation of alpha-pinene took place and that its disappearance was not the result for hydrolysis or any other physicochemical process (e.g., volatilization of the hydrocarbon monoterpenes).

Experiment/ compound

Reactor type

Inoculum

Lag period (h)

Maximum degradation rate (mg l-1h-1)

Normalized degradation rate (h-1)

Experiment 1

α-pinene

CSR1

Unacclimated soil A extract

200

0.029

NM

Experiment 2

α-pinene

Serum tubes

Acclimated (from CSR1)

98

0.63

0.042

The normalized degradation rate is the maximum degradation rate normalized to biomass concentration expressed as volatile suspended solids

CSR 1: continuously-stirred reactor 1

NM: not measured because of lack of accurate biomass data

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, alpha pinene was readily degraded by cultures derived from forest soils.
Executive summary:

In a ready biodegradation study, alpha pinene was tested at concentrations of 0.5-3 mg/L. Forest-soil extract cultures were used as inocula for the experiments conducted first without (experiment 1), then with prior microbial acclimation to the test material (experiment 2). The degradation of the test material was assessed by the determination of the biomass, concentration of the test material and headspace CO2. The test treatments and control (sodium azide, 2.5 g/L) were measured in duplicates. The lack of any substantial change in alpha pinene concentration in the azide-amended control reactor demonstrated that disappearance of the test item in the test reactor was not the result of hydrolysis or any other physicochemical process. The normalised degradation rate in experiment 2 was 0.042 h-1. The maximum degradation rate in experiments 1 and 2 were 0.029 and 0.63 mg/L/h, respectively. The lag period in experiments 1 and 2 were 200 and 98 h, respectively. Under the test conditions, alpha pinene was readily degraded by cultures derived from forest soils.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 301 C. No data on GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
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.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
not specified
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(%ThOD)
Value:
>= 90 - <= 95
Sampling time:
28 d
Remarks on result:
other: Indirect analysis (BOD): 91, 90 and 95%; Direct analysis (GC): 100, 100 and 100%
Validity criteria fulfilled:
yes
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.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 301 C. No data on GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
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.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
not specified
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(%ThOD)
Value:
>= 1 - <= 4
Sampling time:
28 d

Camphene reached 1-4 % of its theoretical BOD after four weeks incubation, so it is not ready biodegradable.

Validity criteria fulfilled:
not specified
Remarks:
(No data on control nor reference substance)
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Camphene reached 1-4 % of its theoretical BOD after four weeks incubation, so it is not ready biodegradable.
Executive summary:

Camphene, present at 100 mg/L, reached 1 -4 % of its theoretical BOD after four weeks incubation with activated sludge inoculum (30 mg/L), using a test according to the Japanese MITI-I test (OECD Guideline 301 C). Under test conditions no biodegradation was observed so biodegradation of this substance is not a fast environmental fate process in water.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
See attached the QMRF and QPRF for the QSAR model.

Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Howard, P.H., Boethling, R.S., Stiteler, W.M., Meylan, W.M., Hueber, A.E., Beauman, J.A., Larosche, M.E. 1992. Predictive model for aerobic biodegradability developed from a file of evaluated biodegradation data. Environ. Toxicol. Chem. 11:593-603.
Specific details on test material used for the study:
SMILES: C(C(CC1C2)C2)(C1(C)C)=C
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

BIOWIN1 (Linear Model): 0.4988

BIOWIN2 (Non-Linear Model): 0.3432

Expert Survey Biodegradation Results:

BIOWIN3 (Ultimate Survey Model): 2.6860 (weeks-months)

BIOWIN4 (Primary Survey Model): 3.4977 (days-weeks)

MITI Biodegradation Probability:

BIOWIN5 (MITI Linear Model): 0.4717

BIOWIN6 (MITI Non-Linear Model): 0.3865

Anaerobic Biodegradation Probability:

BIOWIN7 (Anaerobic Linear Model): -0.0855

Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
The substance D-camphene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)
Executive summary:

The substance D-camphene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
See attached the QMRF and QPRF for the QSAR model.

Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Howard, P.H., Boethling, R.S., Stiteler, W.M., Meylan, W.M., Hueber, A.E., Beauman, J.A., Larosche, M.E. 1992. Predictive model for aerobic biodegradability developed from a file of evaluated biodegradation data. Environ. Toxicol. Chem. 11:593-603.
Specific details on test material used for the study:
SMILES: C(CC1C2)C(C2=C)C1(C)C
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

BIOWIN1 (Linear Model): 0.4988

BIOWIN2 (Non-Linear Model): 0.3432

Expert Survey Biodegradation Results:

BIOWIN3 (Ultimate Survey Model): 2.6860 (weeks-months)

BIOWIN4 (Primary Survey Model): 3.4977 (days-weeks)

MITI Biodegradation Probability:

BIOWIN5 (MITI Linear Model): 0.4717

BIOWIN6 (MITI Non-Linear Model): 0.3865

Anaerobic Biodegradation Probability:

BIOWIN7 (Anaerobic Linear Model): -0.0855

Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
The substance fenchene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)
Executive summary:

The substance fenchene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
See attached the QMRF and QPRF for the QSAR model.

Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Howard, P.H., Boethling, R.S., Stiteler, W.M., Meylan, W.M., Hueber, A.E., Beauman, J.A., Larosche, M.E. 1992. Predictive model for aerobic biodegradability developed from a file of evaluated biodegradation data. Environ. Toxicol. Chem. 11:593-603.
Specific details on test material used for the study:
SMILES: C(C(CC1C2)C1(C)C)(=C2)C
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

BIOWIN1 (Linear Model): 0.4988

BIOWIN2 (Non-Linear Model): 0.3432

Expert Survey Biodegradation Results:

BIOWIN3 (Ultimate Survey Model): 2.6860 (weeks-months)

BIOWIN4 (Primary Survey Model): 3.4977 (days-weeks)

MITI Biodegradation Probability:

BIOWIN5 (MITI Linear Model): 0.4462

BIOWIN6 (MITI Non-Linear Model): 0.3302

Anaerobic Biodegradation Probability:

BIOWIN7 (Anaerobic Linear Model): 0.0285

Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
The substance L-alpha pinene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)
Executive summary:

The substance L-alpha pinene was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)

Description of key information

Weight of evidence. Data from peer reviewed publications: Camphene was found not ready biodegradable (1 -4% %ThOD) using activated sludge in the Japanese MITI test. Alpha pinene was determined to be readily biodegradable in a Japanese MITI test using activated sludge. D-alpha pinene was readily degraded by cultures derived from forest soils.

Weight of evidence. Data from EPI-Suite, EPA (USA) / BIOWIN v4.10 calculation method. All main components of the reaction mass were individually predicted to be not ready biodegradable.

Weight of evidence. Conclusion. The main constituents L-camphene, D-camphene and fenchene which represents a composition higher than 80% of the multi-constituent test substance are calculated to be not ready biodegradable. Only ca. 12% of the multi-component substance was calculated to be readily biodegradable. Furthermore, predictions from EPI-Suite, EPA (USA) / BIOWIN v4.10 calculation method indicate that none of the main components is ready biodegradable.Thus, the test substante has been determined to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Weight of evidence. Data from peer reviewed publication: Camphene, present at 100 mg/L, reached 1 -4 % of its theoretical BOD after four weeks incubation with activated sludge inoculum (30 mg/L), using a test according to the Japanese MITI-I test (OECD Guideline 301 C). Under test conditions no biodegradation was observed so biodegradation of this substance is not a fast environmental fate process in water.

Weight of evidence. Data from peer reviewed publication: 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.

Weight of evidence. Data from peer reviewed publication: In a ready biodegradation study, alpha pinene was tested at concentrations of 0.5-3 mg/L. Forest-soil extract cultures were used as inocula for the experiments conducted first without (experiment 1), then with prior microbial acclimation to the test material (experiment 2). The degradation of the test material was assessed by the determination of the biomass, concentration of the test material and headspace CO2. The test treatments and control (sodium azide, 2.5 g/L) were measured in duplicates. The lack of any substantial change in alpha pinene concentration in the azide-amended control reactor demonstrated that disappearance of the test item in the test reactor was not the result of hydrolysis or any other physicochemical process. The normalised degradation rate in experiment 2 was 0.042 h-1. The maximum degradation rate in experiments 1 and 2 were 0.029 and 0.63 mg/L/h, respectively. The lag period in experiments 1 and 2 were 200 and 98 h, respectively. Under the test conditions, alpha pinene was readily degraded by cultures derived from forest soils.

Weight of evidence. Data from EPI-Suite, EPA (USA) / BIOWIN v4.10 calculation method. All main components of the reaction mass were individually predicted to be not ready biodegradable.

Weight of evidence. Conclusion. The main constituents L-camphene, D-camphene and fenchene which represents a composition higher than 80% of the multi-constituent test substance are calculated to be not ready biodegradable. Only ca. 12% of the multi-component substance was calculated to be readily biodegradable. Furthermore, predictions from EPI-Suite, EPA (USA) / BIOWIN v4.10 calculation method indicate that none of the main components is ready biodegradable.Thus, the test substante has been determined to be not ready biodegradable.