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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001-11-28 to 2002-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): TOYOCAT-RX4
- Substance type: clear yellowish liquid
- Physical state: liquid- Analytical purity: 93.1 % (GC)
- Impurities (identity and concentrations): not reported
- Lot/batch No.: 080401
- Expiration date of the lot/batch: 25 October 2002
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal sewage treatment plant 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands
- Storage conditions: sludge was kept under continuous aeration until further treatment
- Storage length: not specified
- Preparation of inoculum for exposure: before use, the sludge was allowed to settle for 30-90 minutes and the liquid decanted for use as inoculum at the amount of 10 mL/L of mineral medium
- Pretreatment: no
- Concentration of sludge: 4.9 g/L suspended solids in the concentrated sludge (information obtained from the municipal sewage treatment plant)
- Initial cell/biomass concentration: 0.42E+06 cells/L (colony count in inoculum was 4.2E+04 cells/mL (determined on agar plates (diameter 9 cm), containing purified agar (Oxoid 18 g/L) and nutrient broth (Oxoid 25 g/L)), and 10 mL of inoculum was added per L of test medium)
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
* Dilution water: tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridgdes (Milli-Q)
* mineral stock solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.5 gNH4Cl dissolved in 1 L Milli-Q water, pH 7.4 ± 0.2
* mineral stock solution B: 22.50 g MgSO4.7H2O dissolved in 1 L Milli-Q water
* mineral stock solution C: 36.4 g CaCl2.2H2O dissolved in 1 L Milli-Q water
* mineral stock solution D: 0.25 g FeCl3.6H2O dissolved in 1 L Milli-Q water
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium
- Test temperature: 22-23 °C
- pH: 7.6 at start of testing, 7.7-8.1 at end of testing
- pH adjusted: no
- Continuous darkness: yes (brown coloured bottles)

TEST SYSTEM
- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration:
* test substance and inoculum: 2 replicates
* inoculum blank: 2 replicates
* positive control: 1 replicate
* toxicity control: 1 replicate
- Method used to create aerobic conditions: A mixture of oxygen (21 %) and nitrogen (79 %) was led through a bottle, containing 0.5-1 L 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca 30-100 mL/min). The initial suspension of unspiked test medium and inoculum was aerated with this CO2-free air overnight to purge the system of CO2 prior to testing. This CO2-free air was also used for aeration during the test.
- Measuring equipment: CO2-evolution was determined through titration of the remaining Ba(OH)2 with 0.05 M standardized HCl.
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2 were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency: every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day
- Sampling method: the absorber bottle closest to the incubation system was sampled each time, the second and third bottle were moved one position closer to the system and a new bottle was added at the end
- On the 28th day, pH of test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. Bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum
- Positive control: containing reference substance and inoculum
- Toxicity control: containing test substance, reference substance and inoculum
Reference substance:
acetic acid, sodium salt
Test performance:
In the toxicity control more than 25 % degradation occurred within 14 days (based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.
The positive control substance was degraded at least 60% within 9 days.
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
28 d
Details on results:
The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In this 28-day CO2 evolution test (modified Sturm test) using microorganisms from domestic non-adapted sludge, the test substance showed to be not readily biodegradable. Only 17% biodegradation was observed after 28 days. No inhibition of microbial activity was observed and degradation of the reference substance indicated that the test system performance was acceptable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
May 2022
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
EPI Suite v4.1
2. MODEL (incl. version number)
BIOWIN v4.11
The sub-models in BIOWIN v4.11 used for this QSAR analysis, are:
- BIOWIN3
- BIOWIN5
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
BIOWIN3 and BIOWIN5 only require a chemical structure as SMILES as input. All SMILES codes are available in the attached QPRF.
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.
5. APPLICABILITY DOMAIN
All biotransformation products fell within the molecular weight and structural applicability domain of BIOWIN3 and BIOWIN5. More details on this assessment can be found in the attached QPRF.
6. ADEQUACY OF THE RESULT
The main purpose of this QSAR analysis, was to assess the ready biodegradation potential of the predicted biodegradation products of JeffCat DPA (CAS# 63469-23-8) for use in the PBT/vPvB assessment. Overall, the predictions can be considered appropriate for this use.
Reason / purpose for cross-reference:
other: Record describing the biodegradation products of JeffCat DPA (CAS# 63469-23-8) predicted by EAWAG-BBD PPS, for which the BCF is estimated.
Principles of method if other than guideline:
For the degradation products of the substance, the ready biodegradation in water is estimated using the Ready Biodegradability prediction in BIOWIN v4.11, as based on its sub-models BIOWIN3 and BIOWIN5.
GLP compliance:
no
Specific details on test material used for the study:
Ready biodegradability predictions were made for the biodegradation products identified with the EAWAG-BBD Pathway Prediction System.
Oxygen conditions:
aerobic
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
other: Of the 41 predicted transformation products, 14 were predicted to be not readily biodegradable and 27 were predicted to be readily biodegradable

The following predictions for ready biodegradation were made with the BIOWIN v4.11 model:














































































































































































Name



BIOWIN v4.11 Ready biodegradability prediction



NA



 YES



NA



 NO



NA



 NO



1-{[3-(Dimethylamino)propyl]amino}-2-propanol



 NO



2-Hydroxypropanal



 YES



Diisopropanolamine



 YES



3-(Dimethylamino)propanal



 NO



NA



 YES



1,1'-[(3-Aminopropyl)imino]di(2-propanol)



 NO



NA



 NO



3-(Methylamino)propanal



 YES



NA



 YES



1,1'-[(3-Hydroxypropyl)imino]diacetone



 NO



NA



 NO



1-{[3-(Dimethylamino)propyl]amino}acetone



 NO



1-[(2-Hydroxypropyl)amino]acetone



 YES



2-Oxopropanal (Methylglyoxal)



 YES



NA



 YES



3-(dimethylamino)-1-propylamine



 NO



1-amino-2-propanol



 YES



Lactic acid



 YES



3-dimethylaminopropionic acid



 YES



2-Oxopropanoic acid



 YES



NA



 YES



3-Oxopropanoic acid



 YES



NA



 YES



NA



 NO



1-[(3-Aminopropyl)amino]-2-propanol



 YES



3-Aminopropanal



 YES



1-Amino-2-propanone



 YES



NA



 NO



1,1'-Iminodiacetone



 YES



NA



 YES



N-Methyl-β-alanin



 YES



NA



 NO



NA



 YES



(2Z)-3-(Methylamino)acrylonitrile                



 NO



NA



 YES



(2E)-3-[(2-Hydroxypropyl)amino]acrylic acid



 YES



NA



 YES



Methylamine



 YES


Conclusions:
The ready biodegradability of each of the predicted degradation products of Jeffcat DPA was predicted using the QSAR model BIOWIN available in the EPI Suite software. Of the 41 degradation products, 14 were predicted to be not readily biodegradable. The remaining 27 degradation products were predicted to be readily biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May 2016 - 29 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms
Version / remarks:
Stipulated in the Thesting Methods for New Chemical Substances (Japan)
Deviations:
no
GLP compliance:
yes
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:
anaerobic
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:
TEST CONDITIONS
- 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

TEST SYSTEM
- 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
- 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

CONTROL AND BLANK SYSTEM
- 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:
aniline
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%
Parameter:
% degradation (DOC removal)
Value:
1
Sampling time:
28 d
Remarks on result:
other: average value of 3 vessels
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
by BOD method
Value:
1
Sampling time:
28 d
Remarks on result:
other: average value of 3 vessels
Parameter:
% degradation (O2 consumption)
Remarks:
by LC-MS method
Value:
1
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:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the substance 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

One study (Mori, 2016) is selected as key study for endpoint coverage and assigned a Klimisch score of 1. The study is a MITI test carried out according to the OECD guideline 301C. After 28 days, the observed biodegradation was 1%. Based on these results, the substance is regarded as not readily biodegradable according to the OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

A second study (Desmares-Koopmans, 2002) investigated the ready biodegradability of the substance in a 28-day CO2 evolution test (modified Sturm test) using microorganisms from domestic non-adapted sludge and performed according to the OECD Guideline 301 B and EU Method C.4-C. A biodegradation of 17% was observed after 28 days, therefore also concluding that the substance does not meet the ready biodegradability criteria. The study was assigned a Klimisch score of 1 and selected as supporting study.


 


Additionally, a QSAR exercise was performed to identify relevant degradation products of the substance, using the EAWAG-BBD Pathway Prediction System model. 41 degradation products were identified, and these were then assessed for their P (B and T) properties in view of the PBT/vPvB assessment. The biodegradability of each of the degradation products was predicted using the QSAR model BIOWIN available in the EPI Suite software. Of the 41 degradation products, 14 were predicted to be not readily biodegradable. The remaining 27 degradation products were predicted to be readily biodegradable.