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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 June 1992 - 30 June 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP, according to international guideline, test substance defined. Not all test details reported
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Principles of method if other than guideline:
The study was performed in 1992 according to OECD 301B with adaptations for volatile substances (sealed vessel). In 2006 the test method was adopted as OECD 310.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent from an unacclimated activated sludge plant
Details on inoculum:
Secondary effluent from an unacclimated activated sludge plant at URL North.
Inoculum used was 10% by volume of the effluent, filtered through a Whatman filter paper (541) to remove coarse particluate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment to pH 6.5.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:

100 ml medium in 160 ml vessels (hypovials)

The air temperature during the 28d-test period was in the range of 20-23 degrees C

TEST CONDITIONS
- Composition of medium: Medium as proposed in 1988 OECD RIng Test on BIodegradation with deviation: Ferric chloride stock solution contained 0.25 g not 0.2 g. The EDTA stock solution contained 0.4 g and not 0.5 g.
. - Solubilising agent (type and concentration if used): test substance was directly added nwith a high quality delivery syringe. 1.4 microliter of the test substance was added to the test medium, corresponding to 1.037 mg carbon (assuming 100% purity of the test substance)
- Test temperature: 20 - 23 degree C during the 28 day period
- pH: no data for the test medium. The inoculum pH was adjusted to 6.5
- Suspended solids concentration: 10% by volume of activated sludge plant effluent (filtered) --> nominal ca 3 mg/L
- Other perfume ingredients were tested at the same time.


TEST SYSTEM
- Culturing apparatus: sealed vessel, 160 ml (hypovials) containing 100 ml mineral medium inocculated with secundary effluent and the respective test or reference substance. Sealed with butyl rubber septum and an aluminium crimp seal.
- Number of culture flasks/concentration: not reported
- Method used to create aerobic conditions: The head space in each vessel has a volume of 60 ml and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the substance.
- Measuring equipment: Ionics 555 Inorganic carbon Analyser (for CO2 in gas, DIC in liquid medium)
- Test performed in closed vessels due to significant volatility of test substance: yes (OECD 310)
- Other:

SAMPLING
- Sampling frequency: day 3, 7, 10, 14, 16, 21, 24, 28
- Sampling method: First the concentration of CO2 in the headspace gas is determined. Then the seal is broken and the concentration of inorganic carbon in the test medium is determined. The total inorganic carbon in the vessel in calculated and corrected by subtracting the total inorganic carbon produced in a control. Based on this, the extent of mineralisation is determined using the initial organic carbon concentration.

CONTROL AND BLANK SYSTEM
- Inoculum blank: not reported
- Abiotic sterile control: not reported
- Toxicity control: not reported
- Other: no data on reference substances reported

Preliminary study:
not relevant
Test performance:
The study report refers to a reference substance, but data on the reference substance were not included in the report. Data on the blank respiration of the inoculum are also not reported. However, a series of 7 test substances was tested at the same time. The viability and low level of endogenous respiration of the inoculum was demonstrated by the varying degree of biodegradation shown for the test substances, ranging from 3 to 85%.
Therefore the lack of data in the report on the reference substance and the blank respiration does not affect the validity of the study results.
Parameter:
% degradation (CO2 evolution)
Value:
58.8
Sampling time:
14 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
71.4
Sampling time:
28 d
Remarks on result:
other: 95% confidence 68.3 - 74.5 %
Details on results:
The biodegradation curve is attached. It is concluded that D-Limonene is classified as readily (meeting the 10d-window) and ultimately biodegradable.
Results with reference substance:
no information

Day

% biodegradation of D-Limonene

3

25.5

7

29.8

10

60.6

14

58.8

16

64.7

21

71.1

24

62.6

28

71.4

Validity criteria fulfilled:
not specified
Remarks:
The viability of the inoculum is demonstrated in the varying degradation results with the 7 test substances tested at the same time.
Interpretation of results:
readily biodegradable
Conclusions:
The test substance is readily biodegradable, meeting the 10d-window.
Executive summary:

The ready biodegradability of D-Limonene (purity 95%) was assessed in a GLP study conducted according to OECD TG 310 (CO2 production in a sealed vesssel, formerly under OECD 301B). After 28 days, the biodegradation was 71.4% (95% confidence interval 68.3 - 74.5%). Thus the test substance is readily biodegradable.

Due to the wide range of degradation results with other test substances, the viability of the inoculum is demonstrated and the lack of a reference substance does not invalidate the test results. The report does not present all test details and therefore the study is rated with a reliability of 2.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Toxicity control not performed; analytical determinations and biodegradation % at different time intervals not reported
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Method: Test material was evaluated for degradation by cultures derived from coniferous forest soil, diluted and used directly without any prior enrichment.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
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 results:
Experiment 1:
- Maximum degradation rate: 0.044 mg/L/h
- Lag period: 182 h
Experiment 2A (d-Limonene tested individually):
- Maximum degradation rate: 0.40 mg/L/h
- Normalised degradation rate: 0.076 /h
- Lag period: 15 h
Experiment 2B (d-Limonene tested with Terpinene and Terpineolene):
- Maximum degradation rate: 0.137 mg/L/h
- Normalised degradation rate: 0.034 /h
- Lag period: 23 h

The detection of CO2, the increase in biomass concentration and lack of any substantial change in the concentration of terpenes in the azide-amended control reactors demonstrated that biodegradation of d-Limonene 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

d-Limonene

CSR1

Unacclimated soil A extract

182

0.044

NM

Experiment 2Aa

d-Limonene

Serum tubes

Acclimated (from CSR1)

15

0.40

0.076

Experiment 2Bb

d-Limonene

Serum tubes

Acclimated (from CSR1)

23

0.137

0.034

a: Four hydrocarbons terpenes tested individually

b: Three hydrocarbons terpenes tested together

NM: not measured because of lack of accurate biomass data

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
d-Limonene was readily degraded by cultures derived from coniferous forest soils.
Executive summary:

In a ready biodegradation study, d-limonene was tested at concentrations of 0.5-3 mg/L. Both forest-soil extract and enriched cultures were used as inocula for the experiments conducted first without microbial acclimation (experiment 1), then with prior microbial acclimation to the test material (experiment 2A). 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 normalized degradation rate in experiments 2A and 2B were 0.076 and 0.034/h, respectively. The normalized degradation rate was not measured in experiment 1 because of lack of accurate biomass data. The maximum degradation rate in experiments 1, 2A and 2B were 0.044, 0.40 and 0.137 mg/L/h, respectively. The lag period in experiments 1, 2A and 2B were 180, 15 and 23 h, respectively. The total mass of the test material in the control reactor remained unchanged throughout the incubation period.

 

d-Limonene was readily degraded by mixed cultures derived from forest soils.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Toxicity control not performed; analytical determinations and biodegradation % at different time intervals not reported
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Soil-slurry biodegradation assay: the test material was evaluated for degradation by indigenous soil microorganisms.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
Details on inoculum:
Source of inoculum: Collected from horizon A of three different watersheds (number 1, 2 and 27) at the Coweeta Hydrologic Laboratory, Otto, N.C.
Soil 1: consisted of only coniferous trees; pH 4.9; moisture 9.8%; organic carbon 7.0 g/100g soil dw
Soil 2: consisted of only coniferous tress; pH 5.2; moisture 12.6%; organic carbon 5.7 g/100g soil dw
Soil 27: consisted of mixed hardwood trees; pH 4.2; moisture 20 %; organic carbon 11.4 g/100g soil dw

Because of no significant difference in either the adsorption pattern or the biological activity among the three soil samples, only soil 27 was selected for the biodegradation assay.
Initial conc.:
0.6 other: mg/tube
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 results:
- Observed volumetric biodegradation rate (mg/L/h): 0.38
- Observed, soil-normalised biodegradation rate (µg/g/h): 1.9

None

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
d-Limonene was readily degraded by indigenous soil microorganisms.
Executive summary:

In a ready biodegradation study, d-limonene was tested at concentrations of 0.6 mg/tube and the inoculum was 20% (w/v) soil-slurry. The degradation of the test material in test treatments, inoculum blank (soil-slurry samples without any test material) and azide-amended (2.5 g/L) soil-slurry controls were assessed by the determination of the biomass, residue test material and headspace CO2.

 

The observed volumetric biodegradation rate and soil-normalised biodegradation rate were determined to be 0.38 mg/L/h and 1.9 µg/g/h.

 

d-Limonene was readily degraded by indigenous soil microorganisms.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
D-Limonene is one of the two isomers of dipentene. Therefore, data on dipentene can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
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%

None

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
dl-Limonene was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, dl-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% of degradation by biochemical oxygen demand was reached in 14 days.

 

Therefore, dl-limonene was readily biodegradable.

 

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
D-Limonene is one of the main constituents of dipentene multiconstituent (REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE). Therefore, data on dipentene multiconstituent can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
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.
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
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 14: > 60%
- % biodegradation on Day 28 = 80%
Results with reference substance:
- Theoretical oxygen demand (ThOD) = 0.8 mg/mg
- % biodegradation on Day 1: > 10%
- % biodegradation on Day 7: > 60%
- % biodegradation on Day 14 = 83%

Table 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 

 5.1 

 8.0 

 3.9 

 

 8.0 

 5.2 

 7.9 

 3.9 

 Mean (M) 

 7.9 

 5.2 

 8.0 

 3.9 

 14 

 7.8 

 3.0 

 7.7 

 3.4 

 

 7.6 

 3.0 

 7.8 

 3.2 

 Mean (M) 

 7.7 

 3.0 

 7.8 

 3.3 

 21 

 7.7 

 2.8 

 7.8 

 

 

 7.6 

 2.6 

 7.6 

 

 Mean (M) 

 7.7 

 2.7 

 7.7 

 

 28 

 7.6 

 2.4 

 7.5 

 

 

 7.6 

 2.2 

 7.6 

 

 Mean (M) 

 7.6 

 2.3 

 7.6 

 

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

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

Oc: Mineral nutrient solution with only inoculum

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

 

Table 2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, Dipentene (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 

 0 

 0 

 7 

 2.7 

 4.1 

 41 

 76 

 14 

 4.7 

 4.5 

 71 

 83 

 21 

 5.0 

 

 76 

 

 28 

 5.3 

 

 80 

 

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:
Dipentene multiconstituent was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 D and GLP, dipentene multiconstituent 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 60% of biodegradation was reached on Day 14. Hence, the test material met the 14-day window requirement for ready biodegradability. On Day 28, the biodegradation was 80%.

 

The reference material, sodium acetate, reached greater than 60% of biodegradation on Day 6. Dipentene multiconstituent 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, it 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.

 

Therefore, dipentene multiconstituent was readily biodegradable.

Description of key information

d-limonene is readily biodegradable according to the criteria of the OECD 301 guideline. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of d-limonene was assessed in a GLP study conducted according to OECD TG 310 (CO2 production in a sealed vesssel, formerly under OECD 301B). The result was confirmed in two other biodegradation studies conducted with the registered substance.

dl-Limonene and dipentene multiconstituent were also found to be readily biodegradable according to OECD 301 criteria, confirming the results obtained with d-limonene.