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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: Deutsche Einhheitsverfahren DEV H-5
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Instead of testing various dilutions of a waste-water the test substance was tested in 5 concentrations chosen on the basis of the TOD (Theoretical Oxygen Demand).
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, non-adapted
Details on inoculum:
Polyvalent waste-water bacteria collected on February 18, 1981, in the aeration unit of the sewage treatment plant of Rheinfelden (Switzerland).
Duration of test (contact time):
5 d
Initial conc.:
> 1 - < 5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Vessels: 100 mL BOD-flasks
- Test concentrations: Blank, 1, 2, 3, 4, 5 mg/L
- Test medium:
- 8.5 mg KH2PO4
- 21.75 mg K2HPO4
- 33.4 mg NaHPO4
- 1.7 mg NH4CI
- 22.5 mg MgSO4.7H2O
- 27.5 mg CaCl2.2H2O
- 0.25 mg FeCl3.6H2O
- Temperature: 20±1 °C
- Measurement : Oxygen concentration in mg/L with ORION-Electrode Type 97-08

Reference substance:
benzoic acid, sodium salt
Test performance:
No abnormalities were seen during the performance of the test.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
5 d
Details on results:
A BOD5 value of 0 mg O2/ mg test substance was observed at all tested concenterations. The TOD was calculated to be 0.384 mg O2/mg test substance.
Parameter:
BOD5
Value:
0 mg O2/g test mat.
Results with reference substance:
The BOD value for the reference substance (sodium benzoate) was measured with 1.275 mg Oxygen/mg (equal to 95.8 % Biodegradation).
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance is not readily biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Principles of method if other than guideline:
Estimation of results of OECD screening tests on ready biodegradation in water: CATALOGIC (v5.13.1.156) BOD 28 days MITI (OECD 301C) v11.15)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Duration of test (contact time):
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 10
Sampling time:
28 d
Details on results:
Model domain similarity:
- Parametric domain: 100%
- Structural domain: 65% (35% unknown)
- Mechanistic domain: 100%
Interpretation of results:
other: not readily biodegradable (according to OECD criteria)
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
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:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Principles of method if other than guideline:
Prediction of ready biodegradability based on six QSAR models
GLP compliance:
no
Oxygen conditions:
aerobic
Duration of test (contact time):
28 d
Key result
Parameter:
other: biodegradation
Sampling time:
28 d
Remarks on result:
other: Biowin1: Does Not Biodegrade Fast; Biowin2: Does Not Biodegrade Fast; Biowin3: Months; Biowin4: Days-Weeks; Biowin5: Does Not Biodegrade Fast; Biowin6: Does Not Biodegrade Fast; Biowin7: Does Not Biodegrade Fast; Ready Biodegradability Prediction: NO

BIOWIN v4.10 Results

Biowin1 (Linear Model Prediction): Does Not Biodegrade Fast

Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

Biowin3 (Ultimate Biodegradation Timeframe): Months

Biowin4 (Primary Biodegradation Timeframe): Days-Weeks

Biowin5 (MITI Linear Model Prediction): Does Not Biodegrade Fast

Biowin6 (MITI Non-Linear Model Prediction): Does Not Biodegrade Fast

Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

Ready Biodegradability Prediction: NO

 

TYPE

 NUM

Biowin1 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Alkyl substituent on aromatic ring       

 0.0547

 0.0547

Frag

 1 

 Azo group  [-N=N-]                      

 -0.2418

 -0.2418

MolWt

 * 

 Molecular Weight Parameter               

        

 -0.1439

Const

 * 

 Equation Constant                        

        

 0.7475

RESULT  

   Biowin1 (Linear Biodeg Probability)    

 0.4165

 

TYPE

 NUM

Biowin2 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Alkyl substituent on aromatic ring       

 0.5771

 0.5771

Frag

 1 

 Azo group  [-N=N-]                      

 -8.2194

 -8.2194

MolWt

 * 

 Molecular Weight Parameter               

        

 -4.2920

RESULT  

   Biowin2 (Non-Linear Biodeg Probability)

 0.0001

 

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast

A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

 

TYPE

 NUM

Biowin3 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Alkyl substituent on aromatic ring       

 -0.0749

 -0.0749

Frag

 1 

 Azo group  [-N=N-]                      

 -0.3004

 -0.3004

MolWt

 * 

 Molecular Weight Parameter               

        

 -0.6679

Const

 * 

 Equation Constant                        

        

 3.1992

RESULT  

 Biowin3 (Survey Model - Ultimate Biodeg)

 2.1560

 

 

TYPE

 NUM

Biowin4 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Alkyl substituent on aromatic ring       

 -0.0685

 -0.0685

Frag

 1 

 Azo group  [-N=N-]                      

 -0.0528

 -0.0528

MolWt

 * 

 Molecular Weight Parameter               

        

 -0.4361

Const

 * 

 Equation Constant                        

        

 3.8477

RESULT  

  Biowin4 (Survey Model - Primary Biodeg) 

3.2903

 

Result Classification:  5.00 -> hours    4.00 -> days   3.00 -> weeks

(Primary & Ultimate)   2.00 -> months   1.00 -> longer

 

 

TYPE

 NUM

Biowin5 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Azo group  [-N=N-]                      

 -0.0459

 -0.0459

Frag

 1 

 Aromatic-CH3                             

 0.0415

 0.0415

Frag

 2 

 Aromatic-H                               

 0.0082

 0.0164

Frag

 1 

 -CH - [cyclic]                          

 0.0124

 0.0124

MolWt

 * 

 Molecular Weight Parameter               

        

 -0.8992

Const

 * 

 Equation Constant                        

        

 0.7121

RESULT  

 Biowin5 (MITI Linear Biodeg Probability)

-0.1626

 

 

TYPE

 NUM

Biowin6 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Azo group  [-N=N-]                      

-10.6129

-10.6129

Frag

 1 

 Aromatic-CH3                             

 0.3072

 0.3072

Frag

 2 

 Aromatic-H                               

 0.1201

 0.2403

Frag

 1 

 -CH - [cyclic]                          

 -0.1295

 -0.1295

MolWt

 * 

 Molecular Weight Parameter               

        

 -8.7256

RESULT  

Biowin6 (MITI Non-Linear Biodeg Probability)

  0.0000

 

A Probability Greater Than or Equal to 0.5 indicates --> Readily Degradable

A Probability Less Than 0.5 indicates --> NOT Readily Degradable

 

 

TYPE

 NUM

      Biowin7 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

 1 

 Alkyl substituent on aromatic ring       

 -0.1145

 -0.1145

Frag

 1 

 Azo group  [-N=N-]                      

 0.0000

 0.0000

Frag

 1 

 Aromatic-CH3                             

 -0.2573

 -0.2573

Frag

 2 

 Aromatic-H                               

 -0.0954

 -0.1909

Frag

 1 

 -CH - [cyclic]                          

 0.0395

 0.0395

Const

 * 

 Equation Constant                        

        

 0.8361

RESULT  

  Biowin7 (Anaerobic Linear Biodeg Prob)

 0.3129

 

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast

A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

 

Ready Biodegradability Prediction: (YES or NO)

----------------------------------------------

Criteria for the YES or NO prediction: If the Biowin3 (ultimate survey

model) result is "weeks" or faster (i.e. "days", "days to weeks", or

"weeks" AND the Biowin5 (MITI linear model) probability is >= 0.5, then

the prediction is YES (readily biodegradable). If this condition is not

satisfied, the prediction is NO (not readily biodegradable). This method

is based on application of Bayesian analysis to ready biodegradation data

(see Help). Biowin5 and 6 also predict ready biodegradability, but for

degradation in the OECD301C test only; using data from the Chemicals

Evaluation and Research Institute Japan (CERIJ) database.

Interpretation of results:
other: not readily biodegradable

Description of key information

The test substance is not readily biodegradable according OECD criteria.

Key value for chemical safety assessment

Additional information

In Article 13 of Regulation (EC) No 1907/2006, it is laid down that information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI (of the same Regulation) are met. Furthermore according to Article 25 of the same Regulation testing on vertebrate animals shall be undertaken only as a last resort.

According to Annex XI of Regulation (EC) No 1907/2006 (Q)SAR results can be used if (1) the scientific validity of the (Q)SAR model has been established, (2) the substance falls within the applicability domain of the (Q)SAR model, (3) the results are adequate for the purpose of classification and labeling and/or risk assessment and (4) adequate and reliable documentation of the applied method is provided.

For the assessment of the test substance (Q)SAR results were used for aerobic biodegradability in water.The criteria listed in Annex XI of Regulation (EC) No 1907/2006 are considered to be adequately fulfilled and therefore the endpoint(s) sufficiently covered and suitable for risk assessment.

Therefore, further experimental studies on aerobic biodegradability in water are not provided.

Both QSAR models show that the test substance is not readily biodegradable according OECD criteria. The EPI Suite v4.10 (BIOWIN Program v4.10) calculation resulted in "not readily biodegradable". The test substance was within the applicability domain of the model. Additionally, the biodegradation was calculated by using CATALOGIC v5.13.1.156 (BOD 28 days MITI (OECD 301C) v11.15. It resulted in 10 % biodegradation after 28 d. Even though the structural domain was correct with only 65 % (35 % unknown) the results support the overall conclusion in the weight of evidence. Furthermore, none of the predicted metabolites are relevant for the subsequent PBT/vPvB assessment as they are either readily biodegradable or have logKow values << 4 which is indicative of a negligible potential to bioaccumulate.

ID Quantity [%] LogKow Smiles BOD [%]
parent 0.3783 0.4982 Cc1cc2c(cc1N=NC1C(=O)NC(=O)NC1=O)NC(=O)N2 10
metabolite 7.877 0.8346 Cc1cc2c(cc1N)NC(=O)N2 0
metabolite 0.2785 -1.9933 NC1C(=O)NC(=O)NC1=O 65
metabolite 60.79 0.5016 Cc1cc2c(cc1N=NC(C(O)=O)C(O)=O)NC(=O)N2 1
metabolite 6.333 -1.5553 NC(N)=O 81
metabolite 25.12 -0.1608 Cc1cc2c(cc1N=NC(C(N)=O)C(O)=O)NC(=O)N2 3
metabolite 5.728 -0.4342 Cc1cc2c(cc1N=NC(C(N)=O)C(N)=O)NC(=O)N2 6
metabolite 2.501 -4.8137 NC(C(O)=O)C(O)=O 58
metabolite 0.3809 -5.0761 NC(C(N)=O)C(O)=O 40
metabolite 0.4656 -1.0365 OC(=O)C(=O)C(O)=O 90
metabolite 0.2729 -1.6989 NC(=O)C(=O)C(O)=O 53
metabolite 1.008 -1.4027 OC(=O)C=O 100

Moreover, the pigment is practically insoluble and therefore considered to be essentially not bioavailable. Due to the large molecular size, cellular uptake of the pigment is unlikely. Hence intracellular biodegradation, which is the main degradation route of bacteria, is probably low. It can be concluded that the substance is likely to persist in the environment. For this reason no ready or inherent degradation studies are proposed.

Additionally, the weight-of-evidence is supported by the estimation of the BOD5 (CIBA-GEIGY Limited 1981). It was tested according to the German Guideline H-5. In this study concentrations of 1, 2, 3, 4 and 5 mg/L of test substance, a negative and a positive control (sodium benzoate) were tested. No biodegradation was observed during the exposure period of 5 days. The BOD5 for the test substance was 0 mg O2/mg test substance, and the TOD was 0.384 mg O2/mg test substance. The BOD5 for the reference substance was measured at 1.275 mg O2/mg which is equal to 95.8% biodegradation.

In overall conclusion, further tests on biodegradation are scientifically not justified.