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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:
weight of evidence
Study period:
1989-09-25 to 1989-12-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study; all validity criteria were fulfilled
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
On-site sludge sampling was carried out at the following 10 locations in Japan:
Fukogawa city sewage plant (Sapporo-shi Hokkaido); Fukashiba industry sewage plant (Kashima-gun Ibaragi); Nakahama city sewage plant (Osaka-shi Osaka); Ochiai city sewage plant (Shinjuku-ku Tokyo); Kitakami river (Ishinomaki-shi Miyagi); Shinano river (Nishikanbara-gun Niigata); Yoshino river (Tokushima-shi Tokushima); Lake Biwa (Otsu-shi Shiga); Hiroshima bay (Hiroshima-shi Hiroshima); Dookai bay (Kitakyushu-shi Fukuoka)
City sewage: Return sludges from sewage plants were collected.
Rivers, lake and sea: Surface water and surface soil which was in contact with the atmosphere were collected.

Preparation of activated sludge:
- The filtrate of the supernatant of an activated sludge in actual use was mixed with an equal volume of the filtrate of the supernatant of a newly collected sludge and the mixture was cultured at pH 7 ± 1 under the sufficient aeration.

Cultivation:
About 30 minutes after ceasing the aeration to the sludge mixture, supernatant approximately equal to 1/3 of the whole volume was removed. Then an equal volume of 0.1 % synthetic sewage was added to the remaining portion, and the mixture was aerated again. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2 °C.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Each 3 mL of solutions A, B, C and D, which are prescribed in JIS K 0102-1986-21, were made up to 1000 mL with purified water (Takasugi Pharmaceutical Co., Ltd.), and then the pH of this solution was adjusted to 7.0.
- Solubilising agent (type and concentration if used): None
- Test temperature: 25 ± 1 °C
- Aeration of dilution water: No data
- Concentration of activated sludge (suspended solids concentration): 30 mg/L
- Continuous darkness: Yes
- Volume of test solution: 300 mL

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus
- Number of culture flasks/concentration: Test solution (water + test substance) - Single; Test solution (sludge + test substance) - triplicate
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Procedure control: Yes with the reference substance
- Toxicity control: Yes

Measurement and analysis for percentage biodegradation:
- Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus
- Determination of test item by gas chromatography (GC)
Reference substance:
aniline
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Remarks on result:
other: Percentage biodegradation by BOD
Key result
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Remarks on result:
other: Percentage biodegradation of test substance (GC)
Details on results:
Appearances of test solutions:

Appearances of test media in cultivation vessels were as follows.
At the initiation of cultivation
- Water + test substance: The test item was not dissolved, and floating on the surface of water.
- Sludge + test substance: The test item was not dissolved, and floating on the surface of water.

At the completion of cultivation
- Water + test substance: Test substance was dissolved in water.
- Sludge + test substance: Test substance was dissolved in test media. Growth of sludge was not observed.

Degree of degradations after 28 days
Degree of degradation by BOD: 2 %, 0 %, 0 % - mean – 1 %
Degree of degradation by GC: 1 %, 2 %, 1 % - mean – 1 %

The test substance is a mixture of cis and trans-configuration isomer. In this study, concentration of test substance cultivated was represented for cis-isomer as 100 mg/L. Degree of biodegradation was calculated only for cis-isomer. The results showed that the test substance was non-biodegradable.
Results with reference substance:
Degree of degradation of aniline calculated by BOD value was 63 % and 75 % at the 7th and 14th day, respectively. It is concluded that this test condition is valid.

Percentage biodegradation

Percentage biodegradations after 28 days were as follows.

 

Table 5.2.1/1: Percentage biodegradation

 

Method

(Sludge + test item)

Bottle-4

Bottle-5

Bottle-6

Mean

Percentage

biodegradation by

BOD

%

2

0

0

1

Percentage

biodegradation of

test item (GC)

%

1

2

1

1

 

 

Table 5.2.1/2: Calculation table for percentage biodegradation by BOD

 

Bottle No.

TOD (mg)

7th day

14th day

28th day

Mean

Deg.

(%)

BOD

(mg)

Deg.

(%)

BOD

(mg)

Deg.

(%)

BOD

(mg)

Deg.

(%)

1

90.3

57.2

63

70.0

75

75.0

78

1

2

 -

0.1

-

2.6

-

4.7

-

3

97.5

3.3

3

5.1

3

7.0

2

4

97.5

1.4

1

3.3

1

4.6

0

5

97.5

0.3

0

1.1

0

1.2

0

6

97.5

0

-

0

-

0

-

 

Deg. : Percentage biodegradation

Bottle No. 1: Sludge + aniline

Bottle No. 6: Water + test substance

Bottle No. 2: Control blank

Bottle No. 3, 4, 5: Sludge + test substance

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not biodegraded by microorganisms under the present test conditions.
Executive summary:

The ready biodegradability of the test item has been investigated according to the OECD Guideline No. 301C.

A measured volume of inoculated mineral medium (suspended sludge concentration at 30 mg/L), containing a known concentration of test substance (100 mg/L) is stirred in a closed flask at a constant temperature (25 ± 1°C) for up to 28 days. Percentage biodegradation by BOD and determination of test item by gas chromatography (GC) were measured.

 

Aniline was used as a reference compound. Degree of degradation of aniline calculated by BOD value was 63 % and 75 % at the 7th and 14th day, respectively. It is concluded that this test condition is valid.

 

Degree of degradation by BOD: 2 %, 0 %, 0 % - mean: 1 %

Degree of degradation by GC: 1 %, 2 %, 1 % - mean: 1 %

 

The test substance was not biodegraded by microorganisms under the present test conditions.

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Not applicable
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
Remarks:
The value is not an experimental result, however the QSAR model is recommended by the ECHA guidance document on information requirements, is well documented with regard to validation parameters according to OECD principles. Moreover, the substance is fully characterised towards the applicability domain.
Justification for type of information:
1. SOFTWARE
EPISUITE v4.1

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

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC=CC(=O)C1C(C)=CCCC1(C)C

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

5. APPLICABILITY DOMAIN
See attached QMRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Qualifier:
according to
Guideline:
other: Reach Guidance on QSAR - R.6
Deviations:
no
Principles of method if other than guideline:
QSAR, Biowin v4.10. More details are given in QMRF/QPRF attached to the dossier.
GLP compliance:
no
Details on inoculum:
not applicable
Details on study design:
not applicable
Preliminary study:
not applicable
Test performance:
not applicable
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Details on results:
Biowin 1 (linear model prediction) = Does Not Biodegrade Fast
Biowin 2 (non-linear model prediction) = Does Not Biodegrade Fast
Biowin 3 (ultimate biodegradation timeframe) = Weeks-Months
Biowin 4 (Primary biodegradation timeframe) = Days-Weeks
Biowin 5 (MITI linear model prediction) = Not Readily Degradable
Biowin 6 (MITI non-linear model prediction) = Not Readily Degradable
Biowin 7 (Anaerobic model prediction) = Does Not biodegrade Fast
Ready Biodegradability Prediction: NO

See details in "Any other information on results incl. tables".
Results with reference substance:
not applicable

Table 5.2.1/1: Biowin 1 result

TYPE

NUM

Biowin 1

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

MolWt

Const

1

1

*

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Molecular weight parameter

Equation constant

-0.1839

0.0068

-0.1839

0.0068

-0.0916

0.7475

RESULT

Biowin 1 (linear biodeg probability)

0.4789

The fragments present in the molecule are within the applicability domain of the model.

Table 5.2.1/2: Biowin 2 result

TYPE

NUM

Biowin 2

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

MolWt

1

1

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Molecular weight parameter

-1.7232

-0.4530

-1.7232

-0.4530

-2.7307

RESULT

Biowin 2 (non-linear biodeg probability)

0.1303

The fragments present in the molecule are within the applicability domain of the model.

For Biowin 1 and 2: a probability less than 0.5 indicates -> Does Not Biodegrade Fast

Table 5.2.1/3: Biowin 3 result

TYPE

NUM

Biowin 3

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

MolWt

Const

1

1

*

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Molecular weight parameter

Equation constant

-0.2121

-0.0225

-0.2121

-0.0225

-0.4250

3.1992

RESULT

Biowin 3 (survey model – ultimate biodeg)

2.5396

The fragments present in the molecule are within the applicability domain of the model. 

Table 5.2.1/4: Biowin 4 result

TYPE

NUM

Biowin 4

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

MolWt

Const

1

1

*

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Molecular weight parameter

Equation constant

-0.1534

-0.0222

-0.1534

-0.0222

-0.2774

3.8477

RESULT

Biowin 4 (survey model – primary biodeg)

3.3946

The fragments present in the molecule are within the applicability domain of the model. 

For Biowin 3 and Biowin 4: 2.0 = months; 3.0 = weeks; 4.0 = days

Table 5.2.1/5: Biowin 5 result

TYPE

NUM

Biowin 5

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

Frag

Frag

Frag

Frag

MolWt

Const

1

1

4

2

1

3

*

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Methyl [-CH3]

-CH2- [cyclic]

-CH- [cyclic]

-C=CH [alkenyl hydrogen]

Molecular weight parameter

Equation constant

0.0676

0.1177

0.0004

0.0197

0.0124

0.0062

0.0676

0.1177

0.0016

0.0395

0.0124

0.0186

-0.5721

0.7121

RESULT

Biowin 5 (MITI linear biodeg probability)

0.3975

The fragments presents in the molecule are within the applicability domain of the model. 

Table 5.2.1/6: Biowin 6 result

TYPE

NUM

Biowin 6

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

Frag

Frag

Frag

Frag

MolWt

1

1

4

2

1

3

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Methyl [-CH3]

-CH2- [cyclic]

-CH- [cyclic]

-C=CH [alkenyl hydrogen]

Molecular weight parameter

0.3990

0.8334

0.0194

0.2365

-0.1295

0.0285

0.3990

0.8334

0.0777

0.4730

-0.1295

0.0855

-5.5516

RESULT

Biowin 6 (MITI non-linear biodeg probability)

0.2164

The fragments presents in the molecule are within the applicability domain of the model.

For Biowin 5 and Biowin 6: a probability less than 0.5 indicates -> Not readily degradable.

Table 5.2.1/7: Biowin 7 result

TYPE

NUM

Biowin 7

FRAGMENT DESCRIPTION

COEFF

VALUE

Frag

Frag

Frag

Frag

Frag

Frag

Const

1

1

4

2

1

3

*

Carbon with 4 single bonds & no hydrogens

Ketone [-C-C(=O)-C-]

Methyl [-CH3]

-CH2- [cyclic]

-CH- [cyclic]

-C=CH [alkenyl hydrogen]

Equation Constant

-0.3342

-0.3919

-0.0796

-0.1200

0.0395

-0.0735

-0.3342

-0.3919

-0.3183

-0.2400

0.0395

-0.2206

0.8361

RESULT

Biowin 7 (anaerobic linear biodeg probability)

-0.6295

The fragments presents in the molecule are within the applicability domain of the model.

For Biowin 7: 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 Biowin 3 result is "weeks" or faster (i.e. "days", "days to weeks", or "weeks") AND the Biowin 5 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. Biowin 5 and 6 also predict ready biodegradability, but for degradation in the OECD 301C test only; using data from the Chemicals Evaluation and Research Institue Japan (CERIJ) database.

Validity criteria fulfilled:
not applicable
Interpretation of results:
other: Not readily biodegradable
Conclusions:
It is expected that the substance is not readily biodegradable.
Executive summary:

The substance ready biodegradation property was investigated using QSAR approach with Biowin v4.10 plug-in from EPISUITE v4.1 from US EPA.

The Biowin 3 result is "weeks-months" and the Biowin 5 probability is less than 0.5, therefore the prediction is not readily biodegradable. In addition, Biowin 6 also predict not readily biodegradable, corresponding to a degradation in the OECD 301C test. In conclusion, it's expected that the substance is not readily biodegradable.

Description of key information

Weight of evidence approach using experimental study (OECD Guideline 301C, no-GLP) and QSAR data (Biowin v4.10):

Not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

To assess the ready biodegradability of the registered substance, a weight of evidence approach was used using one experimental study and one QSAR data.

The experimental study was performed on the registered substance according to OECD Guideline 301C but without GLP statement. The test substance (100 mg/L) was exposed to activated sewage sludge microorganisms with culture medium in closed bottles at a constant temperature (25 +/- 1°C) for 7, 14 and 28 days. The degradation of the test substance was assessed by the measurement of the percentage biodegradation by BOD and the determination of the test substance by Gas Chromatography (GC). According to the results of this study, the test substance was not biodegraded by microorganisms: mean of 1% biodegradation were observed after 28 days using the two methods of determination (BOD and GC). Thus, the test substance should be regarded as not readily biodegradable according to this test.

The QSAR data was performed on the registered substance. The model used is Biowin v4.10, plug-in from EPISUITE v4.1 from US EPA. This model is recommended by the ECHA guidance document on information requirements and is well documented with regard to validation parameters according to OECD principles. Moreover, the substance is fully characterised towards the applicability domain. The Biowin 3 result is "weeks-months" and the Biowin 5 probability is less than 0.5, therefore the prediction is not readily biodegradable. In addition, Biowin 6 also predict not readily biodegradable, corresponding to a degradation in the OECD 301C test. In conclusion, it's expected that the substance is not readily biodegradable.

In conclusion, as we obtained the same results between the experimental and the predicted data, we can conclude, based on this weight of evidence approach, that the registered substance is not readily biodegradable.