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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
Study period:
January 20, 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
according to
other: Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances
Version / remarks:
according to
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Ten types of sludge were collected from different locations (e.g. surface water of rivers, marshes and inland seas including top soil return, and sludge of sewage disposal plant)
- Storage length: Mixed sewage (activated sludge) was used in testing after 22.5 hours
- Preparation of inoculum for exposure: The ten sludges were mixed at the testing laboratory to form an activated sludge. An unspecified volume of solution was added to the activated sludge that contained glucose, peptone, and monopotassium phosphate dissolved in purified water (pH 7.0 ± 1.0)
- Concentration of sludge: Suspended solid concentration of the activated sludge was 4040 mg/l, based on 14.1. of JIS K 0102-2010. Activated sludge was added to bottles containing the test substance, aniline (control material), and a blank (sludge blank) to give a suspended solid concentration of 30 mg/l
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: A 3 ml solution of A, B, C, and D (see 21. of JIS K 0102-2010) was added to 988 ml purified water, followed by pH adjusted to 7.0 (Takasugi Pharmaceutical Co., Ltd.). 5 L of basic medium was prepared in total
- Test temperature: 25 ±1 °C
- pH adjusted: Yes, to 7.0
- Suspended solids concentration: 2.33 ml of activated sludge was added to bottles that contained 2-iso-butoxyethanol, aniline (control), and a blank (sludge blank) to make a 30 mg/l suspended solid concentration

- Culturing apparatus: Six bottles
- Number of culture flasks/concentration: One bottle contained 297 ml purified water and 3 ml of 10 g/l 2-iso-butoxyethanol to create 100 mg/l test material. Three bottles contained 2.33 ml activated sludge, 294.77 ml culture medium and 3ml of 10.0 g/l 2-iso-butoxyethanol (to create 100 mg/l test material). One bottle was prepared that contained 2.33 ml sludge, 297.77 ml basic medium, and 29.5 μl (30 mg) aniline to create 100 mg/l of the control substance. A final bottle contained 297.77 ml basic culture medium and 2.33 ml activated sludge to create a sludge blank
- Measuring equipment: An unspecified instrument was used to measure oxygen consumption, a total organic carbon analyser was used to measure dissolved organic carbon (DOC), and liquid chromatography mass spectrometry (LC-MS) was carried out for quantitative analysis of the test material. Material was weighed accurately with an electric analytical scale

- Sampling frequency: Biological oxygen demand (BOD) was measured on day 7, 14, 21, and 28

- Inoculum blank: Activated sludge blank that contained 297.77 ml basic culture medium and 2.33 ml activated sludge
- Other control: Aniline with activated sludge was prepared to check the activation level of sludge
Reference substance:
Wako Pure Chemical Industries, Ltd., Lot No. DCP5097
Test performance:
Aniline was used to check the activation level of sludge. As 64.0% of aniline was biodegraded after 14 days and this value is higher than the reference value of 60%, it was concluded that the control results were valid.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: Mean
Details on results:
By day 28, the mean degradability of the test substance based on LC-MS was 84 % and 2-isobutoxyacetic acid was detected as the only transformation product. It is possible that the decrease in pH of the test solution to 5.0 to 5.2 along with the production of 2-isobutoxyacetic acid, which may lower the activity of the microorganism and the biodegradation of the test substance may have caused the BOC to remain at only 14% on average.
Results with reference substance:
Aniline had a reported BOD of 49.5, 61.0, 69.6, and 69.7 mg on day 7, 14, 21, and 28, respectively.

Biodegradability after 28 days

Sludge + test substance





BOD degradability






DOC degradability





Degradability of test substance (LC-MS)






Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
A study was undertaken to determine the potential biodegradability of 2-iso-butoxyethanol in water over a 28-day period (based on O2 consumption). In conclusion, percentage biodegradability of the registered substance was found to be 14 % by day 28 and it can, therefore, be considered to be non-readily biodegradable under the conditions of this test.
Executive summary:

The ready biodegradability of 2-iso-butoxyethanol in water was examined in a 28-day experiment performed in line with the Japanese Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011) and OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)) without deviation. The experiment was compliant with Good Laboratory Practise (GLP). Activated sludge was attained by mixture of ten sources of sludge from a sewage disposal plant and river, marsh, and inland sea surface water. The following treatments were prepared and included purified water + 2-iso-butoxyethanol (n = 1), activated sludge + 2 -iso-butoxyethanol (n = 3), activated sludge + aniline (n = 1), and an activated sludge blank (n = 1). Biochemical oxygen demand (BOD) and dissolved organic carbon (DOC) were measured and 2-iso-butoxyethanol was quantitatively analysed by liquid chromatography mass spectrometry (LC-MS) to determine degradability.

By end-experiment, 2 -iso-butoxyethanol had not been degraded in the water-only treatment. Comparatively, in the 2 -iso-butoxyethanol + activated sludge containers, 14 % of the registered substance had been degraded (based on O2 consumption). A large portion (value not quantified due to unavailable sample) had been converted to 2-iso-butoxyacetate. No other breakdown product was identified. It was reported that the generation of 2-iso-butoxyacetate led to a decrease in pH to 5.0 - 5.2 that in turn decreased microbial activity and resulted in lesser biodegradation of the test substance. Control results were determined to be valid because aniline was biodegraded by 64 % by day 14 (higher than the 60 % reference value), inferring that the sludge was indeed activated. Subsequently, 2-iso-butoxyethanol can be determined to be non-readily biodegradable under the conditons of this test. The 10-day window concept does not apply to the Ministry of International Trade and Industry, Japan (MITI) guideline.

Description of key information

2-Iso-butoxyethanol was determined not to be readily biodegradable in water under the conditions of the test. 14 % biodegradation (based on O2 consumption) of the registered substance was observed by day 28. As the biodegradation is <60% the substance is deemed to be not readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The result obtained for the biodegradation in water screening test was obtained in a key study performed according to Good Laboratory Practise (GLP), Japanese Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011) and OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)) without deviation. The key value has subsequently been assigned Klimisch score of 1 (reliable without restriction).