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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:
20 June 2012 to 19 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of activated sludge: A sample of activated sludge was obtained from Worlingworth sewage treatment works (Suffolk, UK).

- Preparation of inoculum for exposure: At the time of collection, the sludge was sieved (1 mm2) then transported to the laboratory and left to stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.
The concentration of suspended solids in a homogenised sample was determined before the start of the test. Aliquots (10 mL) of the sludge were filtered through dried and pre-weighed Whatman GF/C filters, which were then dried again at approximately 105°C for one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the sludge was then determined and the volume required to give a solids level of 30 mg/L in test cultures was calculated. This was added to bottles one day before test initiation to allow a period of ageing.

Duration of test (contact time):
ca. 28 d
Initial conc.:
9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: activated sludge, predominantly domestic waste
- Additional substrate: no
- Test temperature: 22 ± 2°C
- Aeration of dilution water: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: In this test, a Co-ordinated Environmental Services (CES) Ltd automated respirometer and associated software was used to monitor the cumulative amount of oxygen consumed by the mixtures.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes


Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 1
Sampling time:
10 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 5
Sampling time:
28 d
Details on results:
The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.61 mgO2/500 mL or 62% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 26.00 mgO2/500 mL or 104% at the end of the test on Day 28.
IIn the presence of RIKABINOL HB, degradation of sodium benzoate had achieved 66% by Day 3.
Cumulative levels of oxygen consumption by the controls after 28 days (18.09 and 20.38 mgO2/500 mL, equivalent to 36.18 and 40.76 mgO2/L) were considered to be acceptable for this assay system. These results confirm that RIKABINOL HB was not inhibitory to the activity of the microbial inoculum and that the test was valid.
Mean oxygen consumption in mixtures containing RIKABINOL HB was equivalent to 5% of the ThOD (25 mgO2/500 mL) at the end of the test (Day 28).
Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days of the consumption achieving 10%. Therefore, RIKABINOL HB was not considered to be readily biodegradable under the conditions of this test.

Calculation of theoretical oxyden demand (ThOD)

The ThOD values of the test substance and sodium benzoate were calculated from their empirical formula to be:

RIKABINOL HB:     C15H28O2                   MW = 240.38 ThOD: 2.80 mgO2/mg

Sodium benzoate:     C7H5O2Na                 MW = 144     ThOD: 1.67 mgO2/mg 

The theoretical oxygen content of the mixtures containing the test substance alone and sodium benzoate were calculated to be 25 mgO2/500 mL or 50 mgO2/L.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days of the consumption achieving 10%. Therefore, RIKABINOL HB was not considered to be readily biodegradable under the conditions of this test.

Description of key information

Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days of the consumption achieving 10%.  Therefore, RIKABINOL HB was not considered to be readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of HBPA was assessed (Huntingdon Life Sciences, 2012, Study MOG0015). The procedure was designed to be compatible the EC Method C.4.-D and the OECD test guidelines 301F, and was conducted in compliance with GLP.

HBPA was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO2/L. Two control cultures contained inoculated mineral salts medium alone and one culture contained inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO2/L). An additional mixture containing sodium benzoate and HBPA (both at 50 mgO2/L) was established in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. 

The parameter determined in the test was the cumulative amount of oxygen consumed by mixtures containing HBPA, mineral salts medium, and an inoculum of activated sludge after 28 days of incubation at temperatures nominally in the range 22 ± 2°C.

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.61 mgO2/500 mL or 62% of the ThOD (25 mgO2/500 mL) after 3 days of incubation. In the presence of HBPA, degradation of sodium benzoate had achieved 66% by Day 3. Cumulative levels of oxygen consumption by the controls after 28 days (18.09 and 20.38 mgO2/500 mL, equivalent to 36.18 and 40.76 mgO2/L) were considered to be acceptable for this assay system. These results confirm that HBPA was not inhibitory to the activity of the microbial inoculum and that the test was valid.

Mean oxygen consumption in mixtures containing HBPA was equivalent to 5% of the ThOD (25 mgO2/500 mL) at the end of the test (Day 28).

HBPA was not considered to be readily biodegradable under the conditions of this test.