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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2006 - February 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted on 17th July 1992
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate
EC Number:
Cas Number:
Molecular formula:
ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate
Test material form:

Study design

Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
The inoculum was the secondary effluent collected from a treatment plant in Bangalore, India, receiving predominantly domestic sewage. A fresh sample of effluent was collected from the treatment plant and was kept aerobic during transport. This effluent was allowed to settle for one hour and decanted. The decanted effluent was preconditioned by aerating for 5 days at 20 to 23°C.
The bacterial population in the inoculum was determined as colony forming units (CFU/mL) by diluting the inoculum up to 10e-6 dilution and then plating on nutrient agar plates.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
33.33 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: According to OECD 301 guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 20-24ºC
- pH: 7.4-7.5
- Aeration of dilution water: Mineral medium was aerated before use
- Continuous darkness: diffuse light

- Culturing apparatus: Conical flasks – 5 L capacity each fitted with an aeration tube reaching nearly to the bottom of the flask and an outlet. Test volume was 3 L.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Carbon dioxide free air was passed through the vessels continuously throughout study
- Measuring equipment: Residual hydroxide in the absorber traps was determined by titration with 0.05 M HCl
- Details of trap for CO2: The outlet of each test flask was connected to the inlet of three gas absorption bottles in series, each containing 100 mL of 0.0125 M barium hydroxide solution. The outlet of the last absorption bottle was left open.
- Other:

- Sampling frequency: On day 3, 6, 8, 10, 14, 17, 21, 24, and 29
- Sampling method: The barium hydroxide gas absorber bottle closest to the test vessel was disconnected. The residual concentration of barium hydroxide was determined by titration with HCl. Following the removal of the first gas absorption bottle, the second was connected to the test vessel and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.

- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes
- Reference control: Yes
Reference substance
Reference substance:
other: sodium acetate

Results and discussion

% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
6 d
% degradation (CO2 evolution)
Sampling time:
17 d
% degradation (CO2 evolution)
Sampling time:
29 d
Details on results:
Cumulative CO2 production in the controls was 139.89 mg CO2 in 3L that was typical for this type of test and inoculum source and was within the acceptable range for this assay system.
The degradation of sodium acetate in the presence of the test item had achieved 61.5% of its CO2 after 14 days. These results show that the test item did not cause any inhibitory effect on the test system at this concentration.

BOD5 / COD results

Results with reference substance:
The degradation of sodium acetate was rapid and had achieved 19.1% of its CO2 after 3 days, 88.5% after 14 days and 98.8% after 29 days.

Any other information on results incl. tables

Table 1: Degradation of test/reference item (% ThCO2)

DayTest itemReferenceTest item + reference
Flask 1Flask 2MeanControlToxicity control


Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Under the test condition the test item was biodegraded by 39.3% after 29 days and is assessed to be not readily biodegradable.
Executive summary:

The ready biodegradability of the test item was tested using the CO2 Evolution Test (Modified Sturm Test) according to OECD Guideline No. 301 B and the EC, Part IV, Method C.4-C.
The test item was added to two test vessels at the concentration of 33.33 mg/L of mineral medium. Two control treatments containing only the inoculum, one positive control treatment containing inoculum plus reference standard (sodium acetate) and one toxicity control treatment containing the inoculum plus test item and reference standard were also tested. All the treatments were prepared with inoculum from a secondary effluent treatment plant receiving predominantly domestic sewage.
Treatment mixtures were aerated for 29 days with carbon dioxide free air. The CO2 released by each treatment mixture was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on 3, 6, 8, 10, 14, 17, 21, 24, and 29 days after the initiation of the test by titration.
Sodium acetate had biodegraded by 19.1% at Day 3 and 88.5% by Day 14 in the absence of test item meeting the validity criteria of the test. Test mixture containing sodium acetate and test item had biodegraded by 61.5% at Day 14 showing that the test item was not inhibitory at this concentration.
The cumulative CO2 production by mixtures containing only test item had achieved 39.3% degradation of test item during the treatment period.

The test item had biodegraded by 2.5% by day 3 and by 23.6% at day 14. Based on the pass levels (60% bio-degradation in the 10-day window period), the test item cannot be considered as readily biodegradable. However, it may be considered biodegradable, as there was 39.3% degradation during the treatment period.