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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
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, d-alpha pinene was readily degraded by cultures derived from forest soils.
Executive summary:

In a ready biodegradation study, d-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 d-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, d-alpha pinene was readily degraded by cultures derived from forest soils.

Description of key information

Key study. Data from peer reviewed publication: d-alpha pinene was readily degraded under aerobic conditions at 23ºC by mixed cultures derived from forest soils.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study. In a ready biodegradation study, d-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 d-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, d-alpha pinene was readily degraded by cultures derived from forest soils.