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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:
04-04-2017 - 03-05-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 and river water instead of an effluent/extract/mixture was used as inoculum.
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Qualifier:
according to
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
- Source of inoculum/activated sludge: River water was sampled from the Rhine near Heveadorp, The Netherlands (29-03-2017). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

Reference
*Ginkel CG van and Stroo CA (1992) Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotoxicol Environ Saf 24 319-327.
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 bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Deionized water
Deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.

Nutrients, stocks and administration:
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per 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 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

Test procedure:
Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Test conditions:
The pH of the media was 7.9 at the start of the test. The pH of the medium at day 28 was 7.9 (controls) and 7.8 (test). Temperatures were within the prescribed temperature range of 22 to 24°C.
Reference substance:
acetic acid, sodium salt
Remarks:
purity > 99% Batch No. BCBP8197V
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 1.3 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 87. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
85
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
77
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
45
Sampling time:
7 d
Details on results:
Cedrol - Cedarwood Texas distilled oil was biodegraded by 85% at day 28 in the Closed Bottle test. The time-day window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Cedrol - Cedarwood Texas distilled oil is a mixture of chemicals (UVCB). The biodegradation kinetics (lag period, growth rate, and yield) of the individual chemicals in a mixture are not necessarily same. The biodegradation of a (fragrance) substance consisting of many constituents is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the multi-constituent suggests that the test substance is not readily biodegradable. The time window that can be applied to Closed Bottle tests is therefore not considered applicable to this multi-constituent substance (OECD, 2006), even though it is met. Over 60% biodegradation was achieved in a period of approximately 10 days immediately following the attainment of 10% biodegradation after 1 to 2 days. The test item therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds. Cedrol - Cedarwood Texas distilled oil should as a consequence be classified as readily biodegradable.

reference
*OECD (2006) Revised introduction to the OECD guidelines for testing of chemicals, section 3, Part 1: Principles and strategies related to the testing of degradation of organic chemicals, Paris Cedex, France.
Results with reference substance:
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of Cedrol - Cedarwood Texas distilled oil to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

A biodegradation of 85% and 87% were measured on day 7 and 14, respectively.

Mean (n=2) dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)

Oxygen concentration (mg/L)

 

Ocs

Ot

Oc

Oa

0

8.8

8.8

8.8

8.8

7

8.3

5.6

8.1

3.6

14

7.8

3.7

7.7

3.0

21

7.6

3.0

7.5

 

28

7.6

2.5

7.5

 

Ocs: River water with nutrients and silica gel.

Ot: River water with nutrients, test material (2.0 mg/L) and silica gel.

Oc: River water with nutrients.

Oa: River water with nutrients and sodium acetate (6.7 mg/L).

  

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, CW Texas Cedrol (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

4.5

45

83

14

4.1

4.7

68

87

21

4.6

 

77

 

28

5.1

 

85

 

Validity criteria fulfilled:
yes
Remarks:
See "test performance" for details
Interpretation of results:
readily biodegradable
Remarks:
CW Texas- Cedrol oil is a mixture of chemicals (UVCB), the time window should therefore not be applied to this multi-constituent substance (OECD, 2006)
Conclusions:
Cedrol - Cedarwood Texas distilled oil was biodegraded by 85% at day 28 in the Closed Bottle test and should be classified as readily biodegradable.
Executive summary:

The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with GLP. Cedrol - Cedarwood Texas distilled oil did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Cedrol - Cedarwood Texas distilled oil was biodegraded by 85% at day 28. Cedrol - Cedarwood Texas distilled oil is a mixture of chemicals (UVCB), the time window concept should therefore not be applied to this multi-constituent substance (OECD, 2006) eventhough it is met. Cedrol - Cedarwood Texas distilled oil should be classified as readily biodegradable

Description of key information

The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with GLP. Cedrol - Cedarwood Texas distilled oil did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Cedrol - Cedarwood Texas distilled oil was biodegraded by 85% at day 28. Cedrol - Cedarwood Texas distilled oil is a mixture of chemicals (UVCB), the time window concept should therefore not be applied to this multi-constituent substance (OECD, 2006) eventhough it is met. Cedrol - Cedarwood Texas distilled oil should be classified as readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information