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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
Study period:
From June 09 to July 08, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guideline 301 D under GLP with only minor deviations (ammonium chloride was omitted from the medium without resulting in nitrogen limitation; activated sludge was used as inoculum)
Qualifier:
according to
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. certificate)
Remarks:
2010-07-21
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
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, and control with silica gel) and 6.9 (test)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Concentration of sludge: The sludge was diluted in the BOD bottles at 2 mg DW/L according to van Ginkel and Stroo (1992)
- 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.
- The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. Administration of L-limonene was accomplished by dosing 0.03 g of L-limonene on 10 g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with alumiminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with L-limonene was added to the test bottles.

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:
Acros organics, Geel, Belgium. CAS reg. No. 127-09-3; Purity >99%; Batch/lot number A0206783001
Preliminary study:
No data
Test performance:
The pass level of 60% was reached after 7 to 8 days upon achieving 10% biodegradation. L-Limonene therefore fulfilled the 14-day time window (10-day for other OECD 301 tests) criterion for ready biodegradable compounds. The 14-day window is considered acceptable for the Closed Bottle test because the number of bottles to evaluate the 10-day window causes the test to become too unwieldy (OECD, 1992). L-Limonene is therefore classified as readily biodegradable.
Although the substance was degraded within a time window of 14 days it should be emphasized that time window should not be used as pass/fail criterion for
substances which consists of mixture of compounds (stereoisomers of limonene). The degradation curve is therefore the sum of two growth curves. The time window criterion was developed on the assumption that a single compound is degraded according to a “standard” growth curve in ready biodegradability tests.
Key result
Parameter:
% degradation (O2 consumption)
Value:
85
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 8: > 60%
- % biodegradation on Day 28 = 85%
Results with reference substance:
- Theoretical oxygen demand (ThOD) = 0.8 mg/mg
- % 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 

 4.2 

 8.0 

 3.9 

 

 8.0 

 4.0 

 7.9 

 3.9 

 Mean (M) 

 7.9 

 4.1 

 8.0 

 3.9 

 14 

 7.8 

 2.6 

 7.7 

 3.4 

 

 7.6 

 2.8 

 7.8 

 3.2 

 Mean (M) 

 7.7 

 2.7 

 7.8 

 3.3 

 21 

 7.7 

 2.2 

 7.8 

 

 

 7.6 

 2.5 

 7.6 

 

 Mean (M) 

 7.7 

 2.4 

 7.7 

 

 28 

 7.6 

 2.0 

 7.5 

 

 

 7.6 

 2.0 

 7.6 

 

 Mean (M) 

 7.6 

 2.0 

 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, L-limonene (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 

 3.8 

 4.1 

 58 

76

 14 

 5.0 

 4.5 

 76 

83

 21 

 5.3 

 

 80 

 

 28 

 5.6 

 

 85 

 

Validity criteria fulfilled:
yes
Remarks:
The validity of the test is demonstrated by: an endogenous respiration at Day 28 of 1.0 mg/L; differences of the replicate values at Day 28 < 20%; degradation in reference material of 83% at Day 14 and O2 concentration during the test> 0.5 mg/L.
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, l-limonene was readily biodegradable.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

l-limonene did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. l-limonene was biodegraded 85% at day 28 in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 83% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The pass level of 60% was reached after 7 to 8 days upon achieving 10% biodegradation. L-Limonene therefore fulfilled the 14-day time window (10-day for other OECD 301 tests) criterion for ready biodegradable compounds. Hence, this substance should be classified as readily biodegradable.

Description of key information

Key study: The ready biodegradability of l-limonene was determined in the Closed Bottle test performed according to slightly modified OECD 301D, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The substance was determined to be ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of l-limonene was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. l-Limonene did not cause a reduction in the endogenous respiration and is therefore considered to be non-inhibitory to the inoculum. l-Limonene was biodegraded 85% at day 28. The test is valid as shown by an endogenous respiration of 1.0 mg/L, by the total mineralization of the reference compound, sodium acetate and by oxygen concentrations> 0.5 mg/L in all bottles during the test period.

The pass level of 60% was reached after 7 to 8 days upon achieving 10% biodegradation. L-Limonene therefore fulfilled the 14-day time window criterion for ready biodegradable compounds. Hence, this substance should be classified as readily biodegradable.