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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The source of test organism was activated sludge freshly obtained from a municipal sewage treatment plant: Waterschap de Maaskant, s-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.4 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant) Before use, the sludge was allowed to settle (83 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
29 d
Initial conc.:
12 mg/L
Based on:
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Pre-incubation medium: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles: Test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Positive control: containing reference substance and inoculum (1 bottle). Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with MiIIi-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH), were connected in series to the exit air line of each test bottle.
Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH), with 0.05 M standardized HCI (1 :20 dilution from 1 M HCI (Titrisol® ampul), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1 % solution in ethanol, Merck, Darmstadt, Germany) was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCI (37%, Merck, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
Theoretical CO, production: The theoretical CO2 production was calculated from the molecular formula.
Measurements and recordings: pH- at the start of the test and on the 28th day. Temperature of medium: Continuously in a vessel with Milli-RO water in the same room. Electronic data capture: Observations/measurements in the study were recorded electronically using the following programme:
REES version 1.5 (REES scientific, Trenton, NJ, USA): Temperature.
Data evaluation: Relative degradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production based on the total carbon content of the amount of test material present in the test bottles. A figure of more than 10% degradation was considered as significant. The relative degradation values were plotted versus time together with the relative degradation of the positive control. If applicable, the number of days is calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be 10 days (10-day window), then the test substance is designated as readily biodegradable. Toxicity control: if less than 25% degradation (based on ThC02) occurred within 14 days, the test substance was assumed to be inhibitory. The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in ml of titrant) between the blank Ba(OH)2 traps and fresh Ba(OH)2.
Reference substance:
other: sodium acetate
% degradation (CO2 evolution)
Sampling time:
28 d
Results with reference substance:
In the toxicity control more than 25% degradation occurred within 14 days (33%, based on ThC02). Therefore, the test substance was assumed not to inhibit microbial activity.
Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
2-Pentanone oxime (Methyl propylketoxime) was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Description of key information

The substance is not readily biodegradable according to OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable

Additional information

The biodegradation of the substance was investigated in a study following OECD guideline 301 B (GLP). Activated sludge obtained from a municipal sewage treatment plant receiving predominantly domestic sewage was mixed with an initial concentration of 12 mg/L test substance. The degradation of the test substance was followed by measuring the CO2 evolution of the test suspension. The CO2 production was compared to the results of a blank control. The results obtained for the inhibition control demonstrate that the substance does not inhibit microbial activity. After 28 days, a biodegradation rate of 9% was determined for 2-Pentanone oxime. Thus, the substance is not readily biodegradable according to OECD criteria.