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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:
24 May 2016 - 29 July 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
according to
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms
Version / remarks:
Stipulated in the Thesting Methods for New Chemical Substances (Japan)
GLP compliance:
Specific details on test material used for the study:
- Chemical name: Reaction product of 3-dimethylaminopropylamine and propylene oxide with 1,1'-[(3-dimethylaminopropyl)imino]-bis-2-propanol and 1-[(3-dimethylaminopropyl)(2-hydroxy-1-methylethyl)amino]-2-propanol as main component
- Name of test material (as cited in study report): DMAPA-2PO
- CAS number: CAS 63469-23-8 (1,1'-[(3-(dimethylaminopropyl)imino)]-bis-2-propanol)
- Molecular formula: C11H26N2O2 (main component)
- Molecular weight: 218.3 (main component)
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100 %
- Impurities (identity and concentrations): not reported
- Lot/batch No.: 5Y1024
- Expiration date of the lot/batch: NA
- Stability under test conditions: stable
Oxygen conditions:
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The sludge came from 10 locations within Japan (like surface water and surface soil of rivers, lakes, inland sea and return sludge from sewage plants)
- Method of cultivation: Activated sludge was cultivated for 18.5h after feeding with the synthetic sewage (prepared using glucose, peptone and potassium dihydrogen phosphate dissolved in purified water). pH was adjusted to 7.0 ± 1.0
- Storage conditions: under laboratory conditions as per the Testing Methods for New Chemicals Substances regulation, Japan
- Storage length: Sampling period began in March 2016 however samples were used starting 20 April 2016.
- Preparation of inoculum for exposure: The amount of activated sludge added to the test vessel was 2.40mL (calculated on the basis of concentration of suspended solid in the activated sludge determined in accordance to the Japanese Industrial Standards K 0102-2013 Section 14.1)
- Pretreatment: not specified
- Concentration of activated sludge: 30 mg/L (as concentration of suspended solids)
- Water filtered: no
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Composition of medium: The basal culture medium of 3L was prepared using purified water (in accordance to Japanese Pharmacopoeia by Takasugi Pharmaceutical) which was added to the 3mL aliquots of solutions to prepare 1L of the solution. The pH of this solution was adjusted to 7.0.
- Additional substrate: not specified
- Test temperature: 25°C ± 1.0
- pH: 7.0 ± 1.0
- pH adjusted: yes
- CEC (meq/100 g): not specified
- Aeration of dilution water: not specified
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

- Culturing apparatus: in glass vessels
- Number of culture flasks/concentration: water + test item: 1; sludge + test item: 3; sludge + aniline: 1; control blank: 1;
- Method used to create aerobic conditions: not applicable
- Method used to create anaerobic conditions: using closed system oxygen consumption apparatus
- Measuring equipment: temperature controlled bath with measuring unit (OM3100A) and data sampler (OM7000A) both from Ohkura Electric
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Soda lime no. 1 from Wako Pure Chemical Industries was used as an absorbent for carbon dioxide

- Sampling frequency: BOD was measured continuously during the test incubation period
- Sampling method: closed system oxygen consumption measuring apparatus
- Sterility check if applicable: not specified
- Sample storage before analysis: under laboratory conditions

- Inoculum blank: comprised basal culture medium to which no test item was added
- Abiotic sterile control: comprised only water and test item
- Toxicity control: comprised sludge and aniline

Reference substance:
Preliminary study:
- The appearance of all three solutions namely, water+test item, sludge + test item and control blank were colourless.
- In the sludge + test item vessel, the growth of sludge was not observed.
Test performance:
- Biodegradation by BOD of the test item was observed to be 1%
- Biodegradation by DOC of the test item was also observed to be 1%
- Biodegradation by LC-MS of the test item was observed to be 2%
% degradation (DOC removal)
Sampling time:
28 d
Remarks on result:
other: average value of 3 vessels
Key result
% degradation (O2 consumption)
by BOD method
Sampling time:
28 d
Remarks on result:
other: average value of 3 vessels
% degradation (O2 consumption)
by LC-MS method
Sampling time:
28 d
Remarks on result:
other: average value of 3 vessels
Results with reference substance:
- The percentage biodegradation of aniline by BOD was 74% after 7 days and 92% after 14 days.
- The value of criterion for the difference between extremes of replicate values of percentage biodegradation was <20%
Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The biodegradation of DMAPA-2PO has been studied in an OECD 301C MITI (I) test. Overall the results of this test show that the test substance is not readily biodegradable under the conditions of the test. Results of this study are reliable without restrictions.

Description of key information

Two good quality guideline studies are available. The first study, a MITI test according to OECD 301C, showed results that also indicated no ready biodegradability (Mori, 2016). In this study biodegradation of around 1% after 28 days was observed.

In the second study (Desmares-Koopmans, 2002), a 28-day CO2 evolution test (modified Sturm test) using microorganisms from domestic non-adapted sludge performed according to OECD Guideline 301 B and EU Method C.4-C , a biodegradation of 17% was observed proving that the substance did not meet the ready biodegradability criteria.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information