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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:
1993-05-19 to 1993-07-12
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Scientifically acceptable study performed according to test guidelines similar to OECD 301, and under GLP conditions.
equivalent or similar to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 6950-77-8,9 (Sanseki-Texaco Chemicals Company, Ltd.)
- Analytical purity: 99.12%
- Impurities (identity and concentrations): hydroxyethoxyethylmorpholine 0.78%, dimorpholinoethane 0.05%

- Storage condition of test material: chilled dark place, sealed vessel
- Stability under test conditions: confirmed by preliminary test

- Name of test material (as cited in study report): bis(2-morpholinoethyl)-ether
- Molecular formula: C12H24N2O3
Oxygen conditions:
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sludge from 3 domestic and 1 industrial WWT plant, as well as surface water and surface soil from 3 rivers, 1 lake and 2 estuaries.
- Method of cultivation: 5 L of filtrate of supernatant of activated sludge in use was mixed with 500 mL of the filtrate of the supernatant of a newly collected sludge and the mixture was cultured at pH 7.0 under sufficient aeration (with prefiltered air).
The following procedure was repeated once every day:
1) aeration was ceased for 30 minutes
2) supernatant corresponding to about 1/3 of the whole volume was removed
3) equal volume of dechlorinated water was added
4) mixture was aerated
5) synthetic sewage(*) was added to a concentration of 0.1 % (w/v)
(*) synthetic sewage: glucose, peptone and monopotassium phosphate dissolved in dechlorinated water - each at concentrations of 5 % (w/v) - and adjusted to pH 7.0 using NaOH.
- Age of culture used for inoculation: ca. 1 month
- Preparation of inoculum: no special treatment reported
- Concentration of sludge: 5400 mg/L suspended solids in culture used for inoculation
- Initial cell/biomass concentration: 30 mg/L suspended solids
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
TOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
- Composition of medium: a basal culture medium was prepared according to Japanese Industrial Standards K 0102-1986-21, no details reported
- Test temperature: 25 ± 1 °C
- pH: intended 7.0 ± 1.0
- Adjustment of pH: yes, to 7.0 in basal culture medium (and additionally in the test substance group after addition of sludge and test substance to basal culture medium)
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

- Culturing apparatus: closed system oxygen consumption measuring apparatus (300 mL in volume), content continuously stirred using magnetic stirrer
- Number of culture flasks/concentration:
* water + test substance: n=1 (no sludge added)
* sludge + test substance: n=3
* sludge + aniline: n=1
* control blank: n=1 (only sludge)
- Details of trap for CO2: soda lime

- pH of test solutions in the test substance group and in the abiotic sterile control was measured at the end of testing.
- Sampling frequency for TOC analysis and GC analysis: once, at test termination
- Sampling method:
A. For the TOC analysis 5 mL was centrifuged at 1000xg during 10 min
B. For the GC analysis the other 295 mL were prepared as described under 'details on pretreatment'

- Inoculum blank: yes, only inoculum added to basal culture medium
- Abiotic sterile control: yes, only purified water and test substance added
- Procedure control: yes, using reference substance aniline
Reference substance:
Test performance:
Percentage biodegradations of aniline calculated by the BOD values were 54 % and 69 % at the 7th and the 14th day, respectively, indicating that the test can be considered valid.
% degradation (O2 consumption)
Sampling time:
28 d
% degradation (TOC removal)
theoretical oxygen demand
Sampling time:
28 d
% degradation (test mat. analysis)
Sampling time:
28 d
Details on results:
It can be concluded that the substance is not biodegradable under the test conditions under consideration.
Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
In this 28-day biodegradation test (performed according to a guideline equivalent to OECD guideline 301C - modified MITI test) no substantial biodegradation of the test substance was observed after 28 days of incubation at an initial concentration of 100 mg/L with 30 mg/L suspended solids. For inoculation an activated sludge composed of a mixture of domestic and industrial sewage, surface water and surface soil from different Japanese locations was used. Only 1 to 4% biodegradation was observed at test finalization, depending on the parameter monitored (BOD, TOC removal, or test material analysis using GC).

Description of key information

The key study was performed equivalent to OECD guideline 301C under GLP conditions. Only 1 to 4 % degradation was observed with the test substance after 28 days, depending on the parameter monitored (BOD, TOC removal, or test material analysis using GC), whereas the reference substance (aniline) showed up to 77 % degradation after 28 days. Up to 50 % of a control substance was degraded after 7 days in the presence of the test substance. Therefore, it can be concluded that the test results are valid. In conclusion, a 28-day modified MITI-test showed that the substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two studies describing a ready biodegradability test were identified. The study of BASF (1997) describes a 28 -days DOC die-away test, and the study of Kurume Research Laboratories (1993) concerns a 28 -days modified MITI test. However, the BASF study also claims to be performed according to OECD 302 B (Zahn-Wellens test), which is an inherent biodegradability test, and according to ISO 7827, which is an ultimate biodegradation guideline. Both studies, although using different methods, come to the same conclusion that the test substance is not biodegradable under test conditions. In the BASF study 0 -10 % biodegradation was observed, whereas the study of Kurume Research Laboratories showed 1 to 4 % degradation. Aniline was used as reference substance in either study. As the study of Kurume Research Laboratories is performed according to GLP and allowed a more thorough evaluation of the performed test, this study was rated K1 and assigned as key study.