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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:
2018-01-31
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)
Version / remarks:
1992
Deviations:
yes
Remarks:
Please refer to "Principles of method"
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
Please refer to "Principles of method"
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)
Version / remarks:
1994
Deviations:
yes
Remarks:
Please refer to "Principles of method"
Qualifier:
according to
Guideline:
other: 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.
Version / remarks:
2006
Deviations:
no
Principles of method if other than guideline:
Slight modifications to the guidelines:

a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound)

b) river water instead of an effluent/extract/mixture was used as inoculum.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
- Source of inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (03-01-2018). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
10 bottles containing river water only
10 bottles containing river water and silica gel
10 bottles containing silica gel with test item
6 bottles containing silica gel and the reference substances (sodium actetate)
10 bottles containing silica gel only

- Additional substrate: 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.

- Solubilising agent: not applicable
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 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. The reference substance (sodium acetate) was added to the bottles using a stock solution of 1.0 g/L.

- Test temperature: 22.7 - 23.0 °C
- pH: 8.0
- pH adjusted: no
- Aeration of water: Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.
- Suspended solids concentration: River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD bottles with glass stoppers
- Number of culture flasks/concentration: 10 per group; test item concentration was 2.0 mg/L
- Method used to create aerobic conditions: The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992).

SAMPLING
- Sampling: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: Reference substance (sodium acetate)
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Results with reference substance:
87 % at day 14

Theoretical oxygen demand (ThOD)

The theoretical oxygen demand (ThOD) of the test substance is 2.4 g O2/g test substance. The ThOD of sodium acetate is 0.8 g/g.

Toxicity

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 tert-butyl 2-ethylperoxyhexanoate 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 (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions

The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 8.0 (controls) and 7.9 (test). The temperature ranged from 22.6 to 22.9°C which is within the prescribed temperature range of 22 to 24°C.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 1.0 mg/L at day 28 (Table I). 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 (Table II and Figure). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability

tert-Butyl 2-ethylperoxyhexanoate was biodegraded by 65% at day 28 in the Closed Bottle test (Table II). 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. tert-Butyl 2-ethylperoxyhexanoate is a substance consisting of two chemicals linked together by a peroxide bond. Upon hydrolysis two compounds are formed i.e. 2-ethylhexanoate and tert-butanol. The biodegradation kinetics (lag period, growth rate, and yield) of the individual hydrolysis products are not necessarily same. The biodegradation of the peroxide is an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the peroxide suggests that the test substance is not readily biodegradable. The time window is therefore not considered applicable to tert-butyl 2-ethylperoxyhexanoate (OECD, 2006). tert-Butyl 2-ethylperoxyhexanoate should therefore be classified as readily biodegradable.

Table 1 Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, tert-Butyl peroxyneodecanoate (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

1.5

4.1

31

76

14

2.2

4.7

46

87

21

2.4

 -

50

 -

28

3.1

 -

65

 -

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of this study, the test item was biotically degraded by 65 % after 28 d. Therefore, the test item is considered readily biodegradable.
Executive summary:

In order to assess the biotic degradation of tert-butyl 2-ethylperoxyhexanoate, a ready bio-degradability 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.

tert-Butyl 2-ethylperoxyhexanoate 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. tert-Butyl 2-ethylperoxyhexanoate was biodegraded by 65% at day 28 in the OECD 301 Closed Bottle test. The substance should therefore be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.

Description of key information

Under the conditions of a closed bottle test according to OECD 301D (2018), the test item was biotically degraded by 65 % after 28 d. Therefore, the test item is considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study:

In order to assess the biotic degradation of tert-butyl 2-ethylperoxyhexanoate, a ready bio-degradability 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. tert-Butyl 2-ethylperoxyhexanoate 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. tert-Butyl 2-ethylperoxyhexanoate was biodegraded by 65% at day 28 in the OECD 301 Closed Bottle test. The substance should therefore be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.