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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening test, other
Remarks:
QSAR (BIOWIN)
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2017-11-14
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
Estimation Programs Interface (EPI) Suite for Microsoft Windows, v4.11 (US EPA, 2012)

2. MODEL (incl. version number)
BIOWIN v4.10

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCCCN(CCO)CCO, See also section ‘Test Material’.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
See attached QPRF.

6. ADEQUACY OF THE RESULT
The model is scientifically valid (see attached QMRF). - See attached QPRF for reliability assessment.
Guideline:
other: REACH Guidance on QSARs R. 6
Principles of method if other than guideline:
- Software tool(s) used including version: EpiSuite v. 4.11
- Model(s) used: BIOWIN v. 4.10
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
Based on:
other: see Remarks
Remarks:
not applicable for in silico study
Parameter followed for biodegradation estimation:
not specified
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 1 (Linear Model prediction)
Value:
0.891
Remarks on result:
other: biodegrades fast
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 2 (Non-Linear Model)
Value:
0.929
Remarks on result:
other: biodegrades fast
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 3 (Ultimate Biodegradation Timeframe)
Value:
3.206
Remarks on result:
other: weeks
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 4 (Primary Biodegradation Timeframe)
Remarks on result:
other: days
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 5 (MITI linear Model prediction)
Value:
0.816
Remarks on result:
other: biodegrades fast
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 6 (MITI Non-Linear Model prediction)
Value:
0.888
Remarks on result:
other: biodegrades fast
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
Biowin 7 (Anaerobic model prediction)
Value:
-0.189
Remarks on result:
other: does not biodegrade fast
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks:
overall ready biodegradability prediction
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Details on results:
According to QSAR (Biodegradation Probability Program) calculations with BIOWIN v4.10 (part of EPI-Win, v4.11) for the test compound:
Biowin1 (Linear Model Prediction): Biodegrades Fast (probability p = 0.891) ,
Biowin2 (Non-Linear Model Prediction): Biodegrades Fast (p = 0.929),
Biowin3 (Ultimate Biodegradation Timeframe): Weeks (p = 3.206),
Biowin4 (Primary Biodegradation Timeframe): Days (p = 3.855),
Biowin5 (MITI Linear Model Prediction): Biodegrades Fast (p = 0.816),
Biowin6 (MITI Non-Linear Model Prediction): Biodegrades Fast (p = 0.888),
Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast (p = -0.189).
Overall Ready Biodegradability Prediction: YES.
The test substance (BDEA) is within the applicability domain of BIOWIN since the predictions are based exclusively on sub-structures known to the model.
Remarks on result:
not measured/tested
Conclusions:
The test substance is predicted to be ready biodegradable by the QSAR Biowin v. 4.10 (Epiwin v. 4.11).
Executive summary:

QSAR (Biodegradation Probability Program) calculations with BIOWIN v4.10 (part of EPI-Win, v4.11) for the test compound BDEA:
Biowin1 (Linear Model Prediction): Biodegrades Fast (probability p = 0.891)
 ,


Biowin2 (Non-Linear Model Prediction): Biodegrades Fast (p = 0.929),


Biowin3 (Ultimate Biodegradation Timeframe): Weeks (p = 3.206),


Biowin4 (Primary Biodegradation Timeframe): Days (p = 3.855),


Biowin5 (MITI Linear Model Prediction): Biodegrades Fast (p = 0.816),


Biowin6 (MITI Non-Linear Model Prediction): Biodegrades Fast (p = 0.888),


Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast (p = -0.189).


Overall Ready Biodegradability Prediction: YES.


 


The test substance (BDEA) is within the applicability domain of BIOWIN since the predictions are based exclusively on sub-structures known to the model. This important validity criterion is discussed in ECHA Guidance (2017b, p. 202). According to the ECHA-Guidance on the Application of the CLP Criteria (ECHA 2017a; Section II.2.3.10: Estimation of degradation), if p-values (probability) below 0.5 are estimated by BIOWIN, “…the substance should be regarded as not rapidly biodegradable”. In case of BDEA biodegradation predictions of sub-tools Biowin1 to Biowin6 exceed this trigger value, i.e. p > 0.5). The software predicts “fast” biodegradation (Biowin1,2,5,6) or an elimination within a range of “days” (Biowin4) to “weeks” (Biowin3) for the substance under discussion. This result supports the assigned key value for biodegradation (“inherently biodegradable, not fulfilling specific criteria”).

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (inoculum taken from wwtp treating industrial sewage)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge from BASF sewage works
- Concentration of sludge: 1000 mg/L
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, non-adapted
Duration of test (contact time):
22 d
Initial conc.:
713 mg/L
Based on:
test mat.
Initial conc.:
400 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- incubation: at room temperature (20-25 °C) on a magnetic stirrer, aerated with air (sparging)
- test substance concentration: calculated initial test item concentration: 400 mg/L DOC (=reference point) measured initial test item concentration: 421 mg/L DOC

TEST SYSTEM
- Culturing apparatus: all glass bottles (volume: 5 L; liquid volume: 3000.0 mL)
- Number of culture flasks/concentration: 1
- Measuring equipment: no data available
- Test performed in open system: yes

SAMPLING
- Sampling: 0 h, 3 h, 1 d, 3 d, 7 d, 10 d, 14 d, 21 d, 22 d
- measurements: pH, DOC (dissolved organic carbon)

CONTROL AND BLANK SYSTEM
- Inoculum blank: no (DOC not determined, but a statistically obtained mean value of 17 mg/L DOC (inoculum: activated sludge from the industrial wwtp of BASF) was used to calculate the DOC-elimination)
- Abiotic sterile control: no
- Toxicity control: no
Parameter:
% degradation (DOC removal)
Value:
98
Sampling time:
22 d
Validity criteria fulfilled:
yes
Remarks:
Basic validity criteria are met according to OECD Guideline 302B. However, inoculum from an industrial STP has been used for this study and neither an inoculum blank nor a postive control with reference compound has been conducted.
Interpretation of results:
other: easily eliminated from water mainly due to biodegradation
Conclusions:
Under the conditions of the test, the substance can be easily eliminated from water mainly due to biodegradation.
Executive summary:

The biodegradation behaviour of the substance Butyldiethanolamine was further investigated in a study similar to OECD Guideline 302 B (BASF AG, 1987). A total exposure duration of 22 days and an initial concentration of 400 mg DOC/L (corresponds to 713 mg/L of test substance) was selected for the study design. As inoculum activated sludge (non-adapted) from an industrial STP was selected. Only one replicate with the above indicated concentration was used. The test substance and the inoculum were placed in a glass bottle (total volume: 5 L; liquid volume: 3 L) which was stirred and aerated with air (sparging). pH and DOC (dissolved organic carbon) measurements were performed after 0 h, 3 h, 1 d, 3 d, 7 d, 10 d, 14 d, 21 d and 22 d. The sigmoidal shape of the degradation curve including a lag phase above 3 days indicates that the degradation of the substance was due to biodegradation rather than abiotic elimination processes like adsorption to activated sludge or volatilization. For the test substance, 98 % (based on DOC removal) of biodegradation was observed after 22 d. A reference substance did not serve as positive control.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-07-01 - 2011-07-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study with GLP compliance
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
extended to 42 days
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Cambridge STW, Cowley Road, Cambridge
- Date of collection: 2011-06-30
- Pretreatment: filtered (Whatman No. 54)
- Concentration of sludge: 1 mL/L
Duration of test (contact time):
42 d
Initial conc.:
2.89 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test temperature: 20 - 20.3 °C

TEST SYSTEM
- Culturing apparatus: closed filled bottles
- Number of culture flasks/concentration: 1 (control), 3 (specific substance concentration)
- Measuring equipment: dissolved oxygen meter

SAMPLING
- Sampling frequency: every 7 days (and at day 0)

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
Reference substance:
acetic acid, sodium salt
Preliminary study:
no preliminary study performed
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
42 d
Details on results:
Values are for a test concentration of 2.89 mg/L and a Theoretical Oxygen Demand (ThOD) of 2186 mg O2/g.
Results with reference substance:
75 % degradation after 28 days

Biochemical oxygen demand


The DO measurements and calculated BOD values for the test substance and reference material are given below:







































































































 Incubation period (days)
 07142128
Incubator temperature (°C)20.020.220.220.320.3
Average blank -DO (mg/L)8.658.438.237.927.59
Replicate number11212123123
Test substance -DO (mg/L)8.638.568.498.368.328.127.988.187.837.747.83
Test substance -BOD (mg O2/g)--52-28-52-38-76-28-97-90-59-90
Reference material - DO (mg/L)8.615.345.515.494.874.824.854.484.504.434.27
Reference material - BOD (mg O2/g)-568536503618570564633568581611

 












































 Incubation period
3542
Incubator temperature (°C)20.320.2
Average blank -DO (mg/L)7.857.43
Replicate number1212
Test substance -DO (mg/l)7.727.847.547.57
Test substance -BOD (mg 02/g)38-3-45-55

 


Theoretical oxygen demand


The measured ThOD value was 2186 mg O2/g n-butylaminodiethanol.


 


 


Degradation


The calculated percent degradation values for the test substance and reference material are given below:





































































 Incubation period (days)
 7142128
Replicate number1212123123
Test substance (%)-2-1-2-2-3-1-4-4-3-4
Average degradation (%)-2-2-3-4
Reference material (%)73696479737281737478
Average degradation (%)71727675

 
































 Incubation period
3542
Replicate number1212
Test substance (%)20-2-3
Average degradation (%)1-2
Validity criteria fulfilled:
yes
Remarks:
Validity criteria (reference material degrades by at least 60 % of its ThOD; blank dissolved oxygen loss does not exceed 1.5 mg/L after 28 days) are met.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance is not readily biodegradable. For several reasons the outcome of this study must be interpreted with care. It is commonly accepted knowledge that there are significant differences regarding applicability of the existing methods (OECD guideline 301 series) for testing ready biodegradability. In the ECHA Guidance on information requirements, Chapter R.7b: Endpoint specific guidance; p. 197) is stated that “it is important to recognize that not all of these test guidelines are equally applicable to all types of substances”.
Comprehensive methodological comparisons (UBA, 2017) revealed that within the range of test designs defined in OECD 301 (series A-F) the method applied by Taminco NV (2011) – i.e. OECD 301 D: Closed Bottle Test – has only a low biodegradation potential (i.e. potency), for instance in compare to a study following OECD 301 B (CO2-Evolution test). Potency of OECD 301 D Guideline is rated with (+) whereas test designs following OECD 302 B achieve (++++). Latter Guideline was applied in the study by BASF SE (2014) and revealed biodegradation rates between 50-60 % CO2/ThCO2. Please refer to UBA (2017; Section 3.1.1, Figure 1) for details of this potency-comparison.
Closed conditions, as required in OECD 301 D (Closed-Bottle test) studies can be a problem in regard of biodegradation of some compounds, whereas in the CO2-evolution test (OECD 301B) the inoculum is continuously aerated with CO2-free air. Following ECHA Guidance (2017b; p. 209) it can be concluded that “A ready-biodegradability test is only a screening test, and if that test could not demonstrate that the substance is readily biodegradable then further testing under less stringent test conditions should be considered at the next level”.

Reference:
UBA-Umweltbundesamt (2017): Assessment of environmental persistence: regulatory requirements and practical possibilities – available test systems, identification of technical constraints and indication of possible solutions. German Environment Agency (UBA), Texte 10/2017, Project No. 54429, Report No. (UBA-FB) 002326; ISSN 1862-4804; January 2017
Executive summary:

The biodegradability of the substance n-butylaminodiethanol (CAS 102-79-4) has been tested according to OECD 301D (closed bottle test) in compliance with GLP. The study duration was prolonged from 28 days to 42 days. A test concentration of 2.89 mg/L has been used. Sodium acetate served as reference substance (75 % degradation after 28 days). The inoculum used for the test was the secondary effluent from a predominantly domestic sewage treatment plant.


The test vessels are closed filled bottles containing the test substance dissolved in the test medium. The bottles were then incubated in the dark at 20.0 to 20.3 °C for the duration of the test period. Initial dissolved oxygen (DO) concentrations were measured, using a dissolved oxygen meter.


Further measurements of dissolved oxygen concentration were made on bottles removed after 7, 14, 21, 28, 35 and 42 days. The COD test was carried out following Standard Methods for the Analysis of Water and Wastewater. However, since the structure was available from an Internet source the ThOD was calculated and used in the test. The use of COD, as a reference value for degradation, is less satisfactory than the ThOD as some chemicals are not fully oxidised in the COD test. Incomplete oxidation values will artificially increase the biodegradation value recorded. After 28 days, 0 % of the test substance were biodegraded. After 42 days, biodegradation was not observed either. The values are based on a Theoretical Oxygen Demand (ThOD) of 2186 mg O2/g.


For several reasons the outcome of this study must be interpreted with care.


It is commonly accepted knowledge that there are significant differences regarding applicability of the existing methods (OECD guideline 301 series) for testing ready biodegradability. In the ECHA Guidance on information requirements, Chapter R.7b: Endpoint specific guidance; p. 197) is stated that “it is important to recognize that not all of these test guidelines are equally applicable to all types of substances”.


Comprehensive methodological comparisons (UBA, 2017) revealed that within the range of test designs defined in OECD 301 (series A-F) the method applied by Taminco NV (2011) – i.e. OECD 301 D: Closed Bottle Test – has only a low biodegradation potential (i.e. potency), for instance in compare to a study following OECD 301 B (CO2-Evolution test). Potency of OECD 301 D Guideline is rated with (+) whereas test designs following OECD 302 B achieve (++++).  Latter Guideline was applied in the study by BASF SE (2014) and revealed biodegradation rates between 50-60% CO2/ThCO2. Please refer to UBA (2017; Section 3.1.1, Figure 1) for details of this potency-comparison.


Closed conditions, as required in OECD 301 D (Closed-Bottle test) studies can be a problem in regard of biodegradation of some compounds, whereas in the CO2-evolution test (OECD 301B) the inoculum is continuously aerated with CO2-free air. Following ECHA Guidance (2017b; p. 209) it can be concluded that “A ready-biodegradability test is only a screening test, and if that test could not demonstrate that the substance is readily biodegradable then further testing under less stringent test conditions should be considered at the next level”.


 


Reference:


UBA-Umweltbundesamt (2017): Assessment of environmental persistence: regulatory requirements and practical possibilities – available test systems, identification of technical constraints and indication of possible solutions. German Environment Agency (UBA), Texte 10/2017, Project No. 54429, Report No. (UBA-FB) 002326; ISSN 1862-4804; January 2017

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-08-01 to 2014-10-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Principles of method if other than guideline:
Contact time of the test compound to the inoculum was prolonged to 60 days.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: Not relevant, no test surrogate or analogue material used.
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 activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 2014-08-04 from the aeration tank of the plant.
- Storage length: 3 days (2014-08-04 until 2014-08-07)
- Pretreatment: A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 72 hours at 22 ± 2 °C.
- Preparation of inoculum for exposure: At the day of exposure the suspension was washed one time with drinking water. Subsequently the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water and the concentration of the sludge was adjusted to 6.0 g/L dry weight.
- Concentration of sludge: Aliquots of 7.5 mL were added to the test vessels to obtain an activated sludge concentration of 30 mg/L dry weight.
- Initial cell/biomass concentration:
- Water filtered: No
Duration of test (contact time):
60 d
Initial conc.:
29 mg/L
Based on:
ThOD/L
Initial conc.:
ca. 34 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS:
- Composition of medium: The used mineral medium complies with the test guideline OECD 301B.
It was prepared as follows:

Solution A:
KH2PO4 (8.50 g) + K2HPO4 (21.75 g) + Na2HPO4 * 2 H2O (33.40 g) + NH4Cl (0.50 g);
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B:
CaCl2 * 2 H2O (36.40 g).
The compound was dissolved with deionized water to 1000 mL.

Solution C:
MgSO4 * 7 H2O (22.50 g).
The compound was dissolved with deionized water to 1000 mL.

Solution D:
FeCl3 * 6 H2O (0.25 g)
The compound was dissolved with deionized water to 1000 mL.
15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

- Additional substrate: No
- Solubilising agent (type and concentration if used): Not used
- Test temperature: 22 ± 2 °C,
- pH: The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2,
- pH adjusted: Yes
- CEC (meq/100 g): Not reported
- Aeration of dilution water: At the start of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous sampling day. The test assays were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow rate of approximately 800 mL per hour.
- Suspended solids concentration: Not reported
- Continuous darkness: yes/no
- Other:

TEST SYSTEM:
- Culturing apparatus: The Carbon Dioxide Evolution Test was performed in 2 L incubation vessels. The test volume was 1.5 L.
- Number of culture flasks/concentration: two
- Method used to create aerobic conditions: The aeration was performed with carbon dioxide free air at a flow rate of approximately 800 mL per hour.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: Refer to section "analytics" of this summary
- Test performed in closed vessels due to significant volatility of test substance: Yes, CO2 was trapped
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: The incubation vessels were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes.

SAMPLING:
- Sampling frequency: Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.
- Sampling method: The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount.
- Sterility check if applicable: Not applicable
- Sample storage before analysis: The test assays were prepared at the day of exposure.

CONTROL AND BLANK SYSTEM:
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: Yes
The following test assays were prepared:
2 blank control assays (BC)
2 test substance assays (TS)
1 inhibition control test assay (IH)
1 reference substance assay (RS)

STATISTICAL METHODS:
No statistical procedures required.
Reference substance:
aniline
Preliminary study:
Not performed
Parameter:
% degradation (CO2 evolution)
Value:
> 50 - <= 60
Sampling time:
60 d
Remarks on result:
other: Degree of biodegradation: 50-60 %CO2/ThCO2 after an exposure period of 60 days
Details on results:
The degree of biodegradation was calculated as mean of the values from two test assays at the end of exposure. The required pass level for the inherent biodegradability was not reached. Based on determined rate of biodegradation at the end of exposure the test substance can be evaluated as partly or moderately biodegradable.
Results with reference substance:
A stock solution with 400.9 mg/L aniline was prepared. The reference substance was dissolved in deionized water and the content of TOC in the stock solution was calculated, using the molecular formula of aniline. To adjust the scheduled test concentrations, suitable aliquots (based on the calculation of the TOC content) were added to the test vessels of reference item assay and inhibition control assay.

Results:
Degree of biodegradation of the reference substance (aniline) after 14 days: 68 % CO2/ThCO2.
The test assay of the reference control was already terminated after an exposure period of 28 days without acidification of the test vessel.

Duration of the adaptation phase: approx. 16 days,


Degree of biodegradation of the test substance at the end of the ten-day window: not applicable,


Degree of DOC removal of test substance at the end of exposure: 90-100 %.









































































































































































Table 1: Account of test results: Direct addition of the test substance


 

 



 



Sample



Blank control



Reference substance



Inhibition control



Test substance


 

 



BC 1



BC 2



 



 



TS 1



TS 2


 

Test substance concentration [mg/L]



 



 



33.9



34.1



33.7


 

TOC concentration of the test substance [mg/L]; nominal value



 



 



20



20


 
TOC concentration of the ref. substance [mg/L]; nominal value 

20



20


  
Deionized water [mL/vessell1473137613761473 
Mineral medium, Solutions A-D [mL/vessel]

19.5



19.5



19.5



19.5


 
Inoculum (6 g/L dry matter) [mL/vessel]7.57.57.57.5 
Addition of the test substance [mg/vessel]; nominal value  50.450.4 
Initial weight of the test substance [mg/vessel]  50.951.150.5 
Stock solution reference substance [mL/vessell 9797   
Test volume [mL]1500150015001500 
pH-values at begin the begin of exposure; checked or adjusted7.47.47.47.47.47.4 
DIC-values at begin of exposurea: 0.6a: 0.7     
 b: 0.6b: 0.7 
pH-value at end of exposure7.47.47.07.17.27.2 
Remark: 
Stock solution, initial weight: 400.9 mg/L 
The pH values were measured and adjusted to the required range, if necessary. 
Legend: DIC=Dissolved Inorganic Carbon; a/b =single values 
Validity criteria fulfilled:
yes
Remarks:
The results in this study are consistent with all validity criteria fulfilled. No deviations from the test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.
Interpretation of results:
other: partly or moderately biodegradable
Conclusions:
Degree of observed biodegradation was 50-60 % CO2/ThCO2 after an exposure period of 60 days. Based on the degree of biodegradation at the end of exposure, Butyldiethanolamine was assessed as partly or moderately biodegradable under the conditions of this test. It is crucial to note that the observed biodegradation (50-60 %) was only slightly below the pass level of the CO2-Evolution Test (60 % CO2/ThCO2) as defined in the respective guideline. In OECD (2006) is further explained that “When results of ready biodegradability tests indicate that the pass level criterion is almost fulfilled (i.e. ThOD or DOC slightly below 60 % or 70 %, respectively) such results can be used to indicate inherent biodegradability.”
The same recommendation is given in ECHA Guidance on Information Requirements, Chapter R.7b (ECHA 2017b, Section 7.9.4.1/7.9.4.4). Therein a biodegradability slightly below pass-level criterion is interpreted as “evidence for inherent biodegradability”, amending that for substances that exhibit between 40 and 60 % mineralization in ready biodegradability tests, extensive primary biodegradation is indicated. Formally Butyldiethanolamine was not inherently biodegradable in this test. Nevertheless, the observed substantial biodegradation (50-60 % during 60 days) clearly indicate that the compound is biodegradable and not persistent in sewage treatment plants and in the aquatic compartment.
Executive summary:

The objective of the study was to determine the biodegradability of Butyldiethanolamine by measurement of the formed carbon dioxide under enhanced test conditions. The test was performed according to OECD Guideline 301 B (CO2 -Evolution Test), US-EPA Test Guidelines OPPTS 835.3110, ISO Standard 9439 (Water Quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of released carbon dioxide) as well as EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test). The degree of biodegradation was calculated as mean of the values from two test assays at the end of exposure. Butyldiethanolamine was not inherently biodegradable in this test. Degree of observed biodegradation was 50-60 % CO2/ThCO2 after an exposure period of 60 days. Based on the degree of biodegradation at the end of exposure, Butyldiethanolamine was assessed as partly or moderately biodegradable under the conditions of this test.


It is crucial to note that the observed biodegradation (50-60 %) was only slightly below the pass level of the CO2-Evolution Test (60 % CO2/ThCO2) as defined in the respective guideline. In OECD (2006) is further explained that “When results of ready biodegradability tests indicate that the pass level criterion is almost fulfilled (i.e. ThOD or DOC slightly below 60 % or 70 %, respectively) such results can be used to indicate inherent biodegradability.” The same recommendation is given in ECHA Guidance on Information Requirements, Chapter R.7b (ECHA 2017b, Section 7.9.4.1/7.9.4.4). Therein a biodegradability slightly below pass-level criterion is interpreted as “evidence for inherent biodegradability”, amending that for substances that exhibit between 40 and 60 % mineralization in ready biodegradability tests, extensive primary biodegradation is indicated. Formally Butyldiethanolamine was not inherently biodegradable in this test. Nevertheless, the observed substantial biodegradation (50-60 % during 60 days) clearly indicate that the compound is biodegradable and not persistent in sewage treatment plants and in the aquatic compartment.

Description of key information

Study similar to OECD 302B_98 % degradation after 22 days (based on DOC removal)
OECD 301B_50-60 % CO2/ThCO2 after 60 days
OECD 301D (Closed Bottle test): study duration was prolonged from 28 days to 42 days, test concentration = 2.89 mg/L, After 28 days, 0 % of the test substance were biodegraded. After 42 days, biodegradation was not observed either. The values are based on a Theoretical Oxygen Demand (ThOD) of 2186 mg O2/g.
QSAR (Biodegradation Probability Program) calculations with BIOWIN v4.10: biodegrades fast (BIOWIN 1, 2, 5, 6, 7); ultimate biodegradation Timeframe: weeks (BIOWIN 3); Primary Biodegradation Timeframe: days (BIOWIN 4)

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

Biodegradation of Butyldiethanolamine (CAS 102 -79 -4) was tested according to OECD Guideline 301 B (CO2 -Evolution Test; BASF SE, 2014). Activated sludge from a municipal sewage plant was used for the study. The test was performed under enhanced conditions since an adaption period (approx. 16 days) was implemented prior to the test phase and the study duration was prolonged to 60 days. Hence, the enhanced screening test followed the recommendations given in UBA (2017; sections 3.3.2 and 3.3.3.3). The degree of biodegradation was calculated as mean of the values from two test assays at the end of exposure. Butyldiethanolamine was not readily biodegradable in this test. Degree of observed biodegradation was 50-60 % CO2/ThCO2 after an exposure period of 60 days. Based on the degree of biodegradation at the end of exposure, Butyldiethanolamine was assessed as partly or moderately biodegradable under the conditions of this test.


It is crucial to note that the observed biodegradation (50-60 %) was only slightly below the pass level of the CO2-Evolution Test (60 % CO2/ThCO2) as defined in the respective guideline. In OECD (2006) is further explained that “When results of ready biodegradability tests indicate that the pass level criterion is almost fulfilled (i.e. ThOD or DOC slightly below 60 % or 70 %, respectively) such results can be used to indicate inherent biodegradability.” The same recommendation is given in ECHA Guidance on Information Requirements, Chapter R.7b (ECHA 2017b, Section 7.9.4.1/7.9.4.4). Therein a biodegradability slightly below pass-level criterion is interpreted as “evidence for inherent biodegradability”, amending that for substances that exhibit between 40 and 60 % mineralization in ready biodegradability tests, extensive primary biodegradation is indicated. Formally Butyldiethanolamine was not readily biodegradable in this test. Nevertheless, the observed substantial biodegradation (50-60 % during 60 days) clearly indicate that the compound is biodegradable and not persistent in sewage treatment plants and in the aquatic compartment. UBA (2017; p. 24) summarizes the function and interpretation of such study design as follows: “Enhanced screening tests are not designed for determining ready biodegradability of a test substance but exclusively to allow an evaluation as not being persistent.” In the UN-Guidance Document (UN, 2007; Appendix I, Section 3.3.3) is stated that “a positive result in an inherent test indicates that the test substance will not persist indefinitely in the environment, however a rapid and complete biodegradation can not be assumed. A result demonstrating more than 70 % mineralization [as revealed in BASF AG, 1984] indicates a potential for ultimate biodegradation. A degradation of more than 20 % [compared to 50-60 % as observed in BASF SE, 2014] indicates inherent, primary biodegradation and a result less than 20 % indicates that the substance is persistent”. Hence, biodegradation potential as concluded for BDEA is in accordance with the advice provided by the relevant guidance documents.


 


In a further screening study (BASF AG, 1987) the biodegradation behaviour of the substance Butyldiethanolamine was investigated according to OECD Guideline 302 B (Zahn-Wellens Test, to characterise inherent biodegradation). A total exposure duration of 22 days and an initial concentration of 400 mg DOC/L (corresponds to 713 mg/L of test substance) was selected for the study design. As inoculum activated sludge (non-adapted) from an industrial STP was selected. Only one replicate with the above indicated concentration was used. The test substance and the inoculum were placed in a glass bottle (total volume: 5 L; liquid volume: 3 L) which was stirred and aerated with air (sparging). pH and DOC (dissolved organic carbon) measurements were performed after 0 h, 3 h, 1 d, 3 d, 7 d, 10 d, 14 d, 21 d and 22 d. The sigmoidal shape of the degradation curve including a lag phase above 3 days indicates that the degradation of the substance was due to biodegradation rather than abiotic elimination processes like adsorption to activated sludge or volatilization. For the test substance, 98 % (based on DOC removal) of biodegradation was observed after 22 d. A reference substance did not serve as positive control.


Due to the strong biodegradation observed in this test the pass criterion for inherent biodegradability was clearly fulfilled and inherent biodegradation can be assumed for the substances. Since specific criteria for this study, however, were not fulfilled (no reference substance, inoculum from industrial STP), the substance is considered as inherently biodegradable, not fulfilling specific criteria.


 


The considerable biodegradation observed in the mentioned studies (BASF AG 1987, BASF SE 2014) is confirmed by outcome of QSAR (Biodegradation Probability Program) calculations with BIOWIN v4.10 (part of EPI-Win, v4.11) for the test compound:
Biowin1 (Linear Model Prediction): Biodegrades Fast (probability p = 0.891)
 ,


Biowin2 (Non-Linear Model Prediction): Biodegrades Fast (p = 0.929),


Biowin3 (Ultimate Biodegradation Timeframe): Weeks (p = 3.206),


Biowin4 (Primary Biodegradation Timeframe): Days (p = 3.855),


Biowin5 (MITI Linear Model Prediction): Biodegrades Fast (p = 0.816),


Biowin6 (MITI Non-Linear Model Prediction): Biodegrades Fast (p = 0.888),


Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast (p = -0.189).


Overall Ready Biodegradability Prediction: YES.


The test substance (BDEA) is within the applicability domain of BIOWIN since the predictions are based exclusively on sub-structures known to the model. This important validity criterion is discussed in ECHA Guidance (2017b, p. 202). According to the ECHA-Guidance on the Application of the CLP Criteria (ECHA 2017a; Section II.2.3.10: Estimation of degradation), if p-values (probability) below 0.5 are estimated by BIOWIN, “…the substance should be regarded as not rapidly biodegradable”. In case of BDEA biodegradation predictions of sub-tools Biowin1 to Biowin6 exceed this trigger value, i.e. p > 0.5). The software predicts “fast” biodegradation (Biowin1,2,5,6) or an elimination within a range of “days” (Biowin4) to “weeks” (Biowin3) for the substance under discussion. This result supports the assigned key value for biodegradation (“inherently biodegradable, not fulfilling specific criteria”).


The biodegradability of the substance Butyldiethanolamine has been also tested according to OECD 301D (Closed Bottle test) in compliance with GLP (Taminco NV, 2011). The study duration was prolonged from 28 days to 42 days. A test concentration of 2.89 mg/L has been used. Sodium acetate served as reference substance (75 % degradation after 28 days). The inoculum used for the test was the secondary effluent from a predominantly domestic sewage treatment plant.


The test vessels are closed filled bottles containing the test substance dissolved in the test medium. The bottles were then incubated in the dark at 20.0 to 20.3 °C for the duration of the test period. Initial dissolved oxygen (DO) concentrations were measured, using a dissolved oxygen meter.


Further measurements of dissolved oxygen concentration were made on bottles removed after 7, 14, 21, 28, 35 and 42 days. The COD test was carried out following Standard Methods for the Analysis of Water and Wastewater. However, since the structure was available from an Internet source the ThOD was calculated and used in the test. The use of COD, as a reference value for degradation, is less satisfactory than the ThOD as some chemicals are not fully oxidised in the COD test. Incomplete oxidation values will artificially increase the biodegradation value recorded. After 28 days, 0 % of the test substance were biodegraded. After 42 days, biodegradation was not observed either. The values are based on a Theoretical Oxygen Demand (ThOD) of 2186 mg O2/g.


For several reasons the outcome of this study must be interpreted with care. In principle, when contradictory results in ready biodegradability tests are obtained, the positive results could be considered valid - irrespective of negative results.


It is commonly accepted knowledge that there are significant differences regarding applicability of the existing methods (OECD guideline 301 series) for testing ready biodegradability. In the ECHA Guidance on information requirements, Chapter R.7b: Endpoint specific guidance; p. 197) is stated that “it is important to recognize that not all of these test guidelines are equally applicable to all types of substances”.


Comprehensive methodological comparisons (UBA, 2017) revealed that within the range of test designs defined in OECD 301 (series A-F) the method applied by Taminco NV (2011) – i.e. OECD 301 D: Closed Bottle Test – has only a low biodegradation potential (i.e. potency), for instance in compare to a study following OECD 301 B (CO2-Evolution test). Potency of OECD 301 D Guideline is rated with (+) whereas test designs following OECD 302 B achieve (++++). Latter Guideline was applied in the study by BASF SE (2104) and revealed biodegradation rates between 50-60 % CO2/ThCO2. Please refer to UBA (2017; Section 3.1.1, Figure 1) for details of this potency- comparison.


Closed conditions, as required in OECD 301 D (Closed-Bottle test) studies can be a problem in regard of biodegradation of some compounds, whereas in the CO2-evolution test (OECD 301B) the inoculum is continuously aerated with CO2-free air. Following ECHA Guidance (2017b; p. 209) it can be concluded that “A ready-biodegradability test is only a screening test, and if that test could not demonstrate that the substance is readily biodegradable then further testing under less stringent test conditions should be considered at the next level”. Hence the study according to OECD Guideline 302 B by BASF SE (2014) should be regarded as a reasonable further tier of testing, based on the limitations observed in the test design applied by Taminco NV (2011).  


 


DISCUSSION AND CONCLUSION:


As described above the dataset consists of two experimental studies (BASF AG 1987, BASF SE 2014) and a QSAR calculation (BIOWIN v4.10) with positive result, i.e. moderate to strong biodegradation of BDEA during the exposure period. On the other hand, the study by Taminco NV (2011) revealed a negative outcome due to lack of any biodegradation during the test period. Following the ECHA-Guidance on the Application of the CLP Criteria (ECHA 2017a; Section 4.1.3.2.4.5: Weight of evidence in degradation), where multiple or conflicting datasets exist for a chemical, the most reliable data should be selected first and “subsequently a weight-of-evidence approach followed based on these data”. Due to the methodological limitations of the performed Closed Bottle test (refer to discussion in text section III above), the lack of biodegradation, as observed in this study is not taken into consideration further in this weight-of-evidence approach. This proceeding is in accordance with above mentioned ECHA-Guidance (2017a), since therein is concluded: “…given the conservative nature of ready biodegradability tests, positive results could be used irrespective of negative results when the scientific quality is good and … guideline criteria are fulfilled.”   


The available measured biodegradation results (BASF AG 1987, BASF SE 2014) – supported by outcome of a Biodegradation Probability Program (BIOWIN v.4.10) provide convincing evidence for the assigned key value (“inherently biodegradable, not fulfilling specific criteria”).


 


References:


ECHA- European Chemicals Agency (2017a): Guidance on the Application of the CLP Criteria. Version 4.0, July 2017


ECHA, European Chemicals Agency (2017b): Guidance on information requirements and chemical safety assessment. Chapter R.7b: Endpoint specific guidance. Version 4.0, June 2017


OECD/OCDE (2006): OECD Guidelines for the Testing of Chemicals. Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, Part 1: Principles and Strategies Related to the Testing of Degradation of Organic Chemicals. Adopted: 23 March 2006


 


UBA-Umweltbundesamt (2017): Assessment of environmental persistence: regulatory requirements and practical possibilities – available test systems, identification of technical constraints and indication of possible solutions. German Environment Agency (UBA), Texte 10/2017, Project No. 54429, Report No. (UBA-FB) 002326; ISSN 1862-4804; January 2017


 


UN (United Nations, 2007): United Nations, Annex 9, Guidance on Hazards to the Aquatic Environment.