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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
Study period:
29-Oct-1996 to 26-Feb-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 301B)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted on 17-Jul-1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland)
- Storage conditions: in test medium, aerated with carbon dioxide-free air overnight
- Preparation of inoculum for exposure: the sludge was washed three times with tap water and an amount corresponding to 4 g (± 10 %) dry material per liter was mixed with Sorensen buffer solution (pH 7)
- Concentration of sludge: an appropriate volume of activated sludge (inoculum) was added to test flasks to achieve a final concentration of 30 mg suspended solids per litre
- Water filtered: no; bidistilled water was used for test medium preparation
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
other: total organic carbon (TOC)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline
- Additional substrate: none
- Solubilising agent: none
- Test temperature: 22-23 °C
- pH: 7.5 (measured at the beginning of the test in all test flasks)
- pH adjusted: no
- Aeration of dilution water: carbon dioxide-free air

TEST SYSTEM
- Culturing apparatus: 5 L all-glass amber bottle
- Number of culture flasks/concentration: 2 flasks
- Method used to create aerobic conditions: aerated with carbon dioxide-free air overnight
- Details of trap for CO2 and volatile organics: 2 absorber flasks, the first one containing 300 mL of 0.05 NaOH and the second one containing 200 mL of 0.05 M NaOH were connected in series to the exit air line of each test flask.

SAMPLING
- Sampling frequency: samples were taken on day 2, 5, 7, 9, 12, 14, 19, 23, 28 and 29
- Sampling method: aliquots of 2.0 mL were taken with a pipette from the absorber flask nearest ot the test flask

CONTROL AND BLANK SYSTEM
- Inoculum blank: test medium and inoculum (2 flasks)
- Abiotic sterile control: test item and sterile test medium (1 flask)
- Toxicity control: test item, positive reference item (aniline, 75 mg), test medium and inoculum (1 flask)
- Procedure control: positive reference item (aniline, 75 mg), test medium and incoculum (2 flasks)
- Test medium blank: sterile test medium (1 flask)

STATISTICAL METHODS:
The amount of carbon dioxide produced was calculated from the inorganic carbon concentration and from the volume of the absorbent (0.05 M NaOH) in the two test flasks.
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
1.9
Sampling time:
7 d
Remarks on result:
other: mean (2 flasks)
Parameter:
% degradation (CO2 evolution)
Value:
0.7
Sampling time:
14 d
Remarks on result:
other: mean (2 flasks)
Parameter:
% degradation (CO2 evolution)
Value:
-2.7
Sampling time:
28 d
Remarks on result:
other: mean (2 flasks)
Details on results:
During 28 days incubation the test item degraded by -2.7 %. Thus, under the conditions of this study, the test item was not readily biodegradable.

No inhibitory effect of the test item on the bacteria was observed (-4.2 to 3.4 %) over the 28-day test period. In the abiotic control containing the test item and sterile test medium, practically no abiotic degradation (-4.9 to 6.0 %) was noted. In the toxicity control containing both the test item and the reference compound aniline, no inhibitory effect on the microorganisms was observed.
Results with reference substance:
In the procedure controls, the reference compound aniline was readily biodegraded by an average of 62.5 % after 14 days of exposure in a 10-day window within the 28-day test period; thus confirming suitability of the activated sludge.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Principles of method if other than guideline:
Estimation of ready biodegradation in water using CATALOGIC v5.11.19 BOD 28 days MITI (OECD 301C) v09.13
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: Model calculation
Duration of test (contact time):
28 d
Parameter:
% degradation (O2 consumption)
Value:
9
Sampling time:
28 d

Concomitant predictions :

Not ready degradable

Primary Half Life = 21.98 days

Ultimate Half Life = 6m 25d

- Predicted value (model result): O2 -consumption (BOD) = 0.09 ± 0.0269

Metabolite prediction:

Biodegradation data of metabolites with estimated quantities ≥0.1% after 28 days (prediction by CATALOGIC 301C v.09.13 implemented in OASIS Catalogic v5.11.19)

Quantity [%]

LogKow

Smiles

BOD predicted [%]*

Remarks

Data from OECD QSAR Toolbox**

41.35

8.82

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1

9

parent

No data

1.969

6.95

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(O)=O)c1

3

metabolite

No data

15.56

6.67

CC(=O)c1cc(C(C)(C)CC(C)(C)C)cc(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

2.227

1.87

OC(=O)c1ccccc1

93

metabolite

Readily biodegradable

1.267

7.86

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccc(O)c(O)c2)c1

11

metabolite

No data

0.1302

8.00

CC(C)(C)CC(C)(C)c1cc(N2NC3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1

11

metabolite

No data

13.93

6.47

CC(C)(C)CC(C)(C)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

1.519

5.87

CC(C)(C)CC(C)(c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1)C(O)=O

9

metabolite

No data

6.075

6.24

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1)C(O)=O

9

metabolite

No data

4.517

4.08

CC(=O)c1cc(C(C)(C)CC(C)(C)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

1.047

3.72

CC(=O)c1cc(C(C)(CC(C)(C)C)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

5.082

5.17

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1

1

metabolite

No data

0.2477

4.36

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(O)=O)c1)C(O)=O

2

metabolite

No data

0.9376

3.51

CC(C)(C)CC(C)(c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

1

metabolite

No data

4.044

3.88

CC(C)(CC(C)(C)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

0

metabolite

No data

20.09

1.99

c1ccccc1

5

metabolite

Readily biodegradable

0.1765

2.62

CC(=O)c1cc(C(C)(C)CC(C)(CO)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

0.1765

2.62

CC(=O)c1cc(C(C)(CC(C)(C)C(O)=O)CO)cc(N2N=C3C=CC=CC3=N2)c1O

2

metabolite

No data

0.1701

2.25

CC(=O)c1cc(C(C)(CC(C)(C)CO)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

2

metabolite

No data

0.309

3.71

CC(C)(CC(C)(C)CO)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1

2

metabolite

No data

0.1522

2.05

CC(C)(CC(C)(c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O)CO

2

metabolite

No data

0.158

2.41

CC(C)(CC(C)(CO)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

2

metabolite

No data

0.158

2.41

CC(C)(CC(C)(CO)C(O)=O)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

0.2467

0.09

CC(O)=O

100

metabolite

Readily biodegradable

0.1565

1.03

Oc1ccccc1O

100

metabolite

Readily biodegradable

1.275

-0.14

OC(=O)CC(=CC(O)=O)C(O)=O

78

metabolite

No data

0.1145

2.98

CC(C)(C)CC(C)(c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1)C(O)=O

0

metabolite

No data

0.4936

3.35

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1)C(O)=O

0

metabolite

No data

0.8722

-0.99

OC(C(O)=O)C(O)(CC(O)=O)C(O)=O

89

metabolite

No data

0.1502

0.96

CC(=C)C(C)=O

9

metabolite

No data

0.3215

-2.06

OC(=O)C(C(O)=O)C(O)=O

97

metabolite

No data

0.3813

-2.47

OC(C(O)=O)C(O)=O

90

metabolite

No data

1.761

-1.25

OC(=O)CC(O)=O

100

metabolite

No data

0.3813

-2.46

OC(=O)CC(O)(C(O)=O)C(O)=O

93

metabolite

No data

0.4623

-1.40

OC(=O)C=O

100

metabolite

Readily biodegradable

0.4623

-2.58

OC(=O)CC(=O)C(O)=O

100

metabolite

No data


 

Interpretation of results:
not readily biodegradable
Remarks:
parent substance
Conclusions:
The parent substance is not readily biodegradable. Several metabolites at a quantity >=0.1% could be identified. According to the predicted BOD and (if available) data from the OECD Toolbox some of these metabolites can be regarded as readily biodegradable. However, for the majority of the metabolites a final conclusion about the degradability/persistency cannot be made.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29-Jan-1999 to 03-Mar-1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without detailed documentation (letter report)
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
not specified
Inoculum or test system:
activated sludge (adaptation not specified)
Duration of test (contact time):
28 d
Initial conc.:
ca. 100 mg/L
Based on:
test mat.
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Remarks on result:
other: No. 1
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Remarks on result:
other: No. 2
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Remarks on result:
other: No. 3
Parameter:
% degradation (test mat. analysis)
Remarks:
(HPLC)
Value:
0
Sampling time:
28 d
Remarks on result:
other: Nos. 1, 2 and 3
Details on results:
The test item was non- biodegradable at a nominal concentration of 100 mg/L based on oxygen consumption (3 %) and HPLC analysis data (0 %) when tested over a period of 28 days.

Description of key information

Not readily biodegradable (according to OECD criteria). 

Key value for chemical safety assessment

Additional information

The ready biodegradability of the test item (parent compound) was assessed over a 28 day period by the modified Sturm test (OECD 301 B). The nominal test concentration was 15 mg a.s./L. The test material did not degrade after the 28 day period (-2.7%). Furthermore, a study conducted according to OECD TG 301 C revealed a degradation rate of 1 to 3% (BOD) and 0% (HPLC). These results indicate that the test item cannot be classified readily biodegradable under the conditions of the test. No degradation is expected.

Furthermore, the degradability of the substance was assessed with Catalogic v5.11.19, Catalogic 301 C v09.13 to identify possible degradation products.

Catalogic v5.11.19, Catalogic 301 C v09.13 predicted a BOD of 9%. The model’s applicability domain is divided into three subdomains. (1) the parameter domain, (2) the structural fragment domain and (3) the metabolic domain. The substance is within the logKow, molecular weight and water solubility ranges of the parameter domain. 95.65% of the fragments of the compound were found in correctly predicted training set chemicals. The remaining 4.35% of the fragments were unknown and not present in the training set chemicals. Thus, the compound is outside of the structural fragments domain despite its good fit. The third subdomain – the metabolic domain – investigates if the compound can be successfully mineralized. The substance is within the metabolic domain. In summary, the substance is not within the total domain of the model, however, the prediction is regarded as reliable. Especially as available experimental data on the confirm the predicted result of 9%.

In conclusion, the compound is clearly regarded as not readily biodegradable (by OECD criteria).

In addition to the prediction of the ready biodegradability the identification of degradation products was performed with the Catalogic 301 C model especially in regards to the PBT assessment. As mentioned above, the prediction of the model is regarded as suitable. Furthermore, the predicted metabolic pathways are regarded as reliable as the underlying metabolic transformations can be applied to the present substance/its atom-centered fragments and especially because the substance is within the metabolic domain of the model. The model predicted 35 metabolites at a relevant quantity of0.1% after 28d (Table 1). In addition to the identification of the metabolites Catalogic 301 C also predicted the BOD. Additionally, for metabolites0.1% the OECD QSAR Toolbox v4.0 was used to identify available experimental data to conclude on the degradability of the metabolites beyond their predicted degradation value. The information is also listed in Table 1.

Table 1: Metabolites at a quantity0.1% after 28d and further information about the distribution (logKow) and the degradability (data from the OECD QSAR Toolbox v4.0).

Quantity [%]

LogKow

Smiles

BOD predicted [%]*

Remarks

Data from OECD QSAR Toolbox**

41.35

8.82

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1

9

parent

No data

1.969

6.95

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(O)=O)c1

3

metabolite

No data

15.56

6.67

CC(=O)c1cc(C(C)(C)CC(C)(C)C)cc(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

2.227

1.87

OC(=O)c1ccccc1

93

metabolite

Readily biodegradable

1.267

7.86

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccc(O)c(O)c2)c1

11

metabolite

No data

0.1302

8.00

CC(C)(C)CC(C)(C)c1cc(N2NC3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1

11

metabolite

No data

13.93

6.47

CC(C)(C)CC(C)(C)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

1.519

5.87

CC(C)(C)CC(C)(c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1)C(O)=O

9

metabolite

No data

6.075

6.24

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)c2ccccc2)c1)C(O)=O

9

metabolite

No data

4.517

4.08

CC(=O)c1cc(C(C)(C)CC(C)(C)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

1.047

3.72

CC(=O)c1cc(C(C)(CC(C)(C)C)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

1

metabolite

No data

5.082

5.17

CC(C)(C)CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1

1

metabolite

No data

0.2477

4.36

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(O)=O)c1)C(O)=O

2

metabolite

No data

0.9376

3.51

CC(C)(C)CC(C)(c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

1

metabolite

No data

4.044

3.88

CC(C)(CC(C)(C)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

0

metabolite

No data

20.09

1.99

c1ccccc1

5

metabolite

Readily biodegradable

0.1765

2.62

CC(=O)c1cc(C(C)(C)CC(C)(CO)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

0.1765

2.62

CC(=O)c1cc(C(C)(CC(C)(C)C(O)=O)CO)cc(N2N=C3C=CC=CC3=N2)c1O

2

metabolite

No data

0.1701

2.25

CC(=O)c1cc(C(C)(CC(C)(C)CO)C(O)=O)cc(N2N=C3C=CC=CC3=N2)c1O

2

metabolite

No data

0.309

3.71

CC(C)(CC(C)(C)CO)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1

2

metabolite

No data

0.1522

2.05

CC(C)(CC(C)(c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O)CO

2

metabolite

No data

0.158

2.41

CC(C)(CC(C)(CO)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O)C(O)=O

2

metabolite

No data

0.158

2.41

CC(C)(CC(C)(CO)C(O)=O)c1cc(C(O)=O)c(O)c(N2N=C3C=CC=CC3=N2)c1O

0

metabolite

No data

0.2467

0.09

CC(O)=O

100

metabolite

Readily biodegradable

0.1565

1.03

Oc1ccccc1O

100

metabolite

Readily biodegradable

1.275

-0.14

OC(=O)CC(=CC(O)=O)C(O)=O

78

metabolite

No data

0.1145

2.98

CC(C)(C)CC(C)(c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1)C(O)=O

0

metabolite

No data

0.4936

3.35

CC(C)(CC(C)(C)c1cc(N2N=C3C=CC=CC3=N2)c(O)c(C(C)(C)C(O)=O)c1)C(O)=O

0

metabolite

No data

0.8722

-0.99

OC(C(O)=O)C(O)(CC(O)=O)C(O)=O

89

metabolite

No data

0.1502

0.96

CC(=C)C(C)=O

9

metabolite

No data

0.3215

-2.06

OC(=O)C(C(O)=O)C(O)=O

97

metabolite

No data

0.3813

-2.47

OC(C(O)=O)C(O)=O

90

metabolite

No data

1.761

-1.25

OC(=O)CC(O)=O

100

metabolite

No data

0.3813

-2.46

OC(=O)CC(O)(C(O)=O)C(O)=O

93

metabolite

No data

0.4623

-1.40

OC(=O)C=O

100

metabolite

Readily biodegradable

0.4623

-2.58

OC(=O)CC(=O)C(O)=O

100

metabolite

No data

*Catalogic v5.11.19, Catalogic 301 C v09.13

**Information on the degradability were derived from the OECD QSAR Toolbox v4.0 including the following databases: (1) Biodegradation in soil OASIS, (2) Biodegradation NITE, (3) ECHA CHEM, and (4) ECOTOX.

The OECD QSAR Toolbox identified only 5 metabolites (excluding the parent compound) with experimental data. These compounds can be regarded as readily biodegradable. For the other metabolites no experimental data could be found and only the predicted BOD values are available. According to the BOD values from Catalogic the majority of the metabolites cannot be regarded as readily biodegradable showing BOD values of <20%.

In an overall conclusion, the parent compound cannot be regarded as readily biodegradable. This is supported by Catalogic 301 C v09.13 which predicted a BOD of 9%. The experimental studies according to OECD TG 301 B and C revealed degradation rates of -2,7, 1-3 and 0%, respectively. According to these results the parent compound is not expected to degrade at all. If at all, a slight degree of primary degradation might be assumed. However, the half life is expected to be long. Therefore, and as a worst case, the parent compound is regarded as persistent in the environment. In a subsequent step, Catalogic was used to identify possible degradation products0.1% which might be relevant in regards to the PBT assessment. For five of these metabolites experimental data derived from the databases incorporated in the OECD QSAR Toolbox could be identified which clearly lead to the conclusion that these five metabolites are readily biodegradable and not persistent. For the remaining metabolites only predicted BOD values from Catalogic 301 C v09.13 are available. Especially the smaller metabolites showed high BOD values. For these compounds it can reasonably be assumed that they are not persistent, but rather well biodegradable if not even readily biodegradable. However, the larger metabolites showed BOD values less than 20%. For these metabolites a concluding assessment about the persistency cannot be made. Therefore, these metabolites are regarded as persistent in the environment. 

 

 

The QSAR predictions have been conducted in compliance with Regulation (EC) No 1907/2006:

 

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 CAS 70321-86-7 (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.