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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:
2010-02-01 to 2010-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted 17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A smaple of activated sludge was obtained from Worlingworth sewage treatment plant (Suffolk,UK). At the time of collection, the sludge was sieved (1mm2) then transported to the laboratory and left 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.
- Preparation of inoculum for exposure: 10 mL aliquots of the sludge were filtered through dried and preweighted Whatman GF/C filters, which were then dried again at 105°C for ca. one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids content of the sludge was determined and the volume to give a solids level of 30 mg/L in test cultures calculated. This was added to the flasks four days before test initiation to allow a period of aging.
-
Duration of test (contact time):
28 d
Initial conc.:
4.808 g/L
Based on:
test mat.
Initial conc.:
50 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium containing 85 mg potassium dihydrogen phosphate per L, 217.5 mg di-Potassium hydrogen phosphate per L, 334 mg di-Sodium monohydrogen phosphate dihydrate per L, 22.5 mg magnesium sulphate hexahydrate per L, 36.4 mg calcium chloride dihydrate per L and 0.25 Iron(III) chloride hexahydrate per l.
- Test temperature: 21.3°C to 22.2°C
- pH: 7.3 - 7.7
- pH adjusted: yes
- Suspended solids concentration: 30 mg/L


TEST SYSTEM
- Culturing apparatus: Amber glass culture bottles
- Number of culture flasks/concentration: 2 blank controls, 2 test material, 1 reference, 1 ATU control, 1 ATU test
- Measuring equipment: each bottle was fitted with an electrolytic cell (containing the electrolyte, 1M
copper sulfate solution, and the CO2 adsorber, 5 mL of 2M potassium hydroxide) and
connected to the respirometer
- Details of trap for CO2 : 5 mL of 2M potassium hydroxide
- The cultures were connected to the CO2 absorber, sealed and placed in a water bath to equilibrate. The cells were connected to the computer-controlled system and the test was initiated.
A record of the cumulative oxygen demand made by each cell was printed at, typically,
hourly intervals.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral salts medium
- Toxicity control: Allylthiourea

Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
ca. 10
Sampling time:
1 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
3 d
Parameter:
% degradation (O2 consumption)
Value:
114
Sampling time:
28 d
Results with reference substance:
The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.3 mgO2/500 mL or 61% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 22.5 mgO2/500 mL or 90% by Day 28. Cumulative levels of oxygen consumption by the controls after 28 days (10.8 and 10.6 mgO2/500 mL, equivalent to 21.6 and 21.2 mgO2/L) were considered to be acceptable for this assay system. These results confirm that the test was valid.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Xylanase, batch PPQ30189 is readily biodegradable according to OECD 301F.
Executive summary:

The ready biodegradability of Xylanase, PPQ 30189 was assessed in accordance with EC Directive 440/2008, C.4-D, ‘Determination of Ready Biodegradability, Manometric respirometry’ and OECD Procedure 301F ‘Ready Biodegradability, Manometric Respirometry Test’, adopted 17 July 1992.

The main biodegradability test was preceded by chemical oxygen demand (COD) analysis as the theoretical oxygen demand of the test substance could not be calculated.

Xylanase, PPQ 30189 was added to two flasks containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO2/L. Control groups comprised two cultures containing inoculated mineral salts medium alone. Allylthiourea (11.6 mg/L) was added to one control culture and to one culture containing the test substance in order to assess nitrification processes by the test substance. The test system comprised of an automated system for oxygen (O2) generation and the cultures were stirred and held in a thermostatically controlled water bath.

The COD of Xylanase, PPQ 30189 was found to be 0.13 mgO2/mg.

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.3 mgO2/500 mL or 61% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 22.2 mgO2/500 mL or 90% by Day 28. Cumulative levels of oxygen consumption by the controls after 28 days (10.8 and 10.6 mgO2/500 mL, equivalent to 21.6 and 21.2 mgO2/L) were considered to be acceptable for this assay system.

Mean oxygen consumption in mixtures containing Xylanase, PPQ 30189 was equivalent to 10% of the theoretical value (25 mgO2/500 mL) after approximately 1 day, 62% after 3 days and 114% at the end of the test (Day 28). The degradation of the test substance beyond 100% was related to the high levels of nitrogen contained in the test substance.

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 Xylanase, PPQ 30189 was considered to be readily biodegradable under the conditions of this test.

Description of key information

The enzyme was found to be readily biodegradable according to OECD 301F.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information