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Environmental fate & pathways

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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
The source of test organisms 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 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):
28 d
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Reference Substance: A solution of sodium acetate was prepared by dissolving 801.5 mg in Milli-RO water and making this up to a total volume of 200 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg/l sodium acetate (12 mg TOC/I).
- Composition of medium: Stock Solutions of mineral components A) 8.50 g KH2P04 21.75 g K2HP04 67.20 g Na2HP04.12H20 0.50 g NH4CI dissolved in Milli-Q water and made up to 1 litre, pH 7.4 +/- 0.2 B) 22.50 g MgS04.7H20 dissolved in Milli-Q water and made up to 1 litre. C) 36.40 g CaCI2.2H2O dissolved in Milli-Q water and made up to 1 litre. D) 0.25 g FeCI3.6H20 dissolved in Milli-Q water and made up to 1 litre. -Mineral Medium- 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
- Test temperature: Continuously in a vessel with Milli-RO water in the same room. The temperature recorded in a vessel with water in the same room varied between 21.3 and 22.9C.
- pH: Measured at the start of the test and on the 28th day.
- pH adjusted: yes with 1 M HCL
- Aeration of dilution water: yes
- Continuous darkness: yes- test vessel 2 litre all glass brown coloured bottles
- Preincubation 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.
- 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 Milli-RO water, resulting in the mineral medium described before. Three C02-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2, were connected in series to the exit air line of each test bottle.
TEST SYSTEM
- Number of culture flasks/concentration: Test suspension: containing test substance and inoculum (2 bottles, lnoculum blank: containing only inoculum (2 bottles); Positive control: containing reference substance and inoculum ( I bottle); Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions: Synthetic Air (CO2< 1 ppm). A mixture of oxygen (21%) and nitrogen (79%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba (OH)2 solution to trap C02 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

SAMPLING
- Sampling frequency: 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 C02-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 C02-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 HCl(37%, Merck, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO, present in the test suspension. The final titration was made on day 29. The theoretical C02 production was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes

STATISTICAL METHODS: The Theoretical CO2 production was calculated from the molecular formula. ThC02, expressed as mg C021mg test substance, that can be generated by a test substance was calculated as follows: ThCO2= No. of carbon atoms in test substance x Molecular weight C02/ molecular weight test substance The first step in calculating the amount of C02, produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2, produced by a test material is determined by the difference (in ml of titrant) between the experimental and blank Ba(OH)2 traps. The amount of 0.05 N HCI titrated is converted into mg of CO2, produced: mgCO2= 0.05 x ¿ ml HCl titrated/ 2 x 44 = 1.1 x ¿ ml HCl titrated Relative degradation values were calculated from the cumulative C02 production relative to the total expected C02 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.
Reference substance:
other: Sodium Acetate
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
28 d
Details on results:
The ThCO2 of OS 1600 was calculated to be 2.05 mg CO2/mg. The ThC02 of Sodium Acetate was calculated to be 1.07 mg CO2/mg. The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of OS 1600. In the toxicity control more than 25% degradation occurred within 14 days (32% based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.
The positive control substance was degraded by at least 60% (63%) within 14 days. Therefore, reference substance considered to be valid.

The positive control substance was degraded by at least 60% (63%) within 14 days.

The difference of duplicate values for % degradation of OS 1600 was always less than 20.

The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (64.5 mg CO2 per 2 litres of medium, corresponding to 32.3 mg/l).

The IC content of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the TC. Since the test medium was prepared using tap water purified by reverse osmosis (Milli-RO water, carbon levels < 500 ppb) the inorganic carbon content was less than 5% of the total inorganic carbon content (mainly coming from the test substance 12 mg TOC/l).

Since all criteria for acceptability of the test were met, this study was considered valid.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
OS 1600 attained 1% biodegradation within 28 days, therefore, it is considered not readily biodegradable.
Executive summary:

The ready biodegradability test was performed in accordance with OECD Guideline 301B and EU Method C4 -C using the carbon dioxide (CO2) evolution test (modified Strum test). The substance was tested in duplicate at 12 mg TOC/L (43 mg/2 litres). The ThCO2 of OS1600 was calculated to be 2.05 mg CO2/mg. Preparation was as much as possible performed under yellow light and/or dimmed light conditions. The test solutions with microbial and mineral components were continuously stirred during the test (28 days) to ensure optimal contact. The relative degradation values based on CO2 consumption calculated from the measurements performed during the test period revealed no significant degradation of OS1600 (1% biodegradation within 28 days). In the toxicity control, the test substance was found not to inhibit microbial activity. The study was considered to be valid. In conclusion, the test item designated as not readily biodegradable.

Description of key information

OS 1600 attained 1% biodegradation within 28 days, therefore, it is considered not readily biodegradable. A ready biodegradation test was also performed on MPKO, the hydrolytic product of OS1600. It was also considered to be not readily biodegradable (4-12% degradation within 28 days).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Key study: The ready biodegradability test was performed in accordance with OECD Guideline 301B and EU Method C4 -C using the carbon dioxide (CO2) evolution test (modified Strum test) (GLP study). Sewage was exposed to 12 mg TOC/L (43 mg/2 litres) of test substance under aerobic conditions for 28 days. The relative degradation values based on CO2 consumption calculated from the measurements performed during the test period revealed no significant degradation of OS1600 (1% biodegradation within 28 days). The study was considered valid. In conclusion, the test item designated as not readily biodegradable.

Supporting study: The ready biodegradability test was performed with the analogue substance MPKO in accordance with OECD Guideline 301B and EU Method C4 -C using the carbon dioxide (CO2) evolution test (modified Strum test) (GLP study). Sewage was exposed to 12 mg TOC/L (40 mg/2 litres) of MPKO under aerobic conditions for 28 days. The relative degradation values based on CO2 consumption calculated from the measurements performed during the test period revealed 4 and 12% degradation within 28 days. The study was considered to be valid. In conclusion, the test item was designated as not readily biodegradable.