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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II; Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was deconated. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ration of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain concentration equivalent to 4 g (+/- 10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weigh basis). Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
test mat.
Initial conc.:
15.1 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask (except for the abiotic control and the abiofic control blank), 90 mL of activated sludge inoculum was added.
For the abiotic control and the abiotic control blank the untreated test medium was poisoned with mercury dichloride at a concentration of 10 mg/L (3 mL of a stock solution containing 10 g HgCl2/L filled up to 3 liter test medium).
The test media were aerated with C02-free air overnight to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of test item were directly weighed into the test flasks. For dosage of the reference item, a stock solution containing 771 mg sodium benzoate per 100 mL test water was prepared. From this a 10 mL aliquot was withdrawn and added to the corresponding test flasks. No emulsifiers or solvents were used.
The test flasks were made up to a volume of three liter with test water (previously purged with C02-free air). Two absorber flasks, the first one containing 300 mL of 0.05 M NaOH and the second one containing 200 mL of 0.05 M NaOH, were connected in series to the exit air line of each test flask.
Because of its light sensitivity the test item was handled in a dark room with red light. The test suspensions were kept in amber flasks.

- Test vessel: 5-liter all-glass amber bottles
- CO2-free air: Air was led through a bottle containing about 750 mL of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30-100 mL/min.
- Number of culture flasks/concentration: 2

- Sampling:
Samples were taken on Day 2, 5, 7, 9, 12, 14, 19, 23, 27, 28 and 29. Additionally, samples were taken on Day 30from both absorber flasks of the toxicity control for data verification.
On each sampling day, an aliquot of 2.0 mL was withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask on Day 14 and at the end of the exposure period on Day 28. On Day 28, the pH was measured in all test flasks. Next, 1 mL of concentrated HCl was added to each test flask and the flasks were aerated overnight to drive off any residual CO2 present. On day 29, a sample of both absorber flasks was withdrawn and anayzed for IC to determine residual CO2 which was present in the tests suspensions on Day 28. Samples of the second NaOH abosrber flask were analyzed in order to correct for any carry over of CO2.

Reference substance:
benzoic acid, sodium salt
sodium benzoate
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: mean value
Details on results:
The CO2 production of the test item in the test media was in the range of the inoculum controls.
Consequently, the test substance was found to be not biodegradable under the test conditions within 28 days.
Results with reference substance:
The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, the reference item was readily degraded by an average of 80% by exposure Day 14, thus confirming suitability of the activated sludge.

Abiotic control:

In the abiotic control containing the test item and pisoned test medium, no significant abiotic degradation (< 10%) was noted at the end of the 28 -day exposure period.

Biodegradation in the toxicity control:

The percent biodegradation in the toxicity control containing both the test item and the reference item was calculated based on the total carbon content (TOC) of the test item and the reference item.

The biodegradation rate in the toxicity control showed a slightly inhibited course of biodegradation over the 28 -day exposure period compared to the two procedure controls containing the reference item only. Within 14 days of exposure, a biodegradation rate of 21% was observed. Between 14 and 28 days of exposure, the biodegradation rate remained practically unchanged in the rang of 21 -22%.

According to the test guidelines, the test item had a slightly inhibitory effect on activated sludge microorganisms because the biodegradation rate in the toxicity control was slightly below 25% within 14 days of exposure.

Driving off of CO2:

Maximum 2.1 mg C/L was found in the absorber flasks. Consequently, only negligible amounts of residual CO2 were present in the test solutions or suspensions at the end of the test.

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
The test substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.
Executive summary:

In this guideline (OECD 301B) study conducted to GLP standards, the test material (EC 438-340-0) was determined to not be readily biodegradable.

Description of key information

Study conducted to recognised testing guidelines with GLP.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

In a ready biodegradability test according to OECD guideline 301B,a maximum CO2 evolution of -7.4% after 28d was observed (RCC Ltd. 2002b). This result is supported by the information from a MITI test (Institute of Ecotoxicology Ltd., Japan 2002), OECD 301C, biodegradation: < 1%).

The substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.