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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:
14.05.1997 - 16.06.1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The sludge is collected, in the mineral medium and kept aerobic until being used on the same day.
Dry weight of suspended solids: 6.136 g/L
To obtain a concentration of 30 mgll (dry weight) in a 250 ml flask, 1.22 mL of sludge is needed (inoculum).
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
nominal concentration
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Solution A+B+C+D (see attachment)
- Test temperature: 22°C
- pH: 7.4 ± 0.2
- pH adjusted: yes

TEST SYSTEM
- Culturing apparatus: flasks
- Number of culture flasks/concentration: 2
- Measuring equipment: respirometer
Reference substance:
benzoic acid, sodium salt
Test performance:
- Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.
- At the end of the test period (normally 28 days), the pH of each flask is measured again.
Key result
Parameter:
% degradation (O2 consumption)
Value:
69
Sampling time:
28 d
Details on results:
See attachment.
Key result
Parameter:
ThOD
Value:
2.86 other: mg O2/mg
Results with reference substance:
Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified and the test is considered as valid.

The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test item on the

micro-organisms at the test concentration. On the contrary, biodegradation of the test substance starts much earlier (day 4) in presence of sodium benzoate.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance undergoes on an average 69 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 17-20 after a long lag phase (adaptation of the inoculum) and reaches a mean value of 68 % at the end of the 10-day window (days 17 to 27).
Thus, the test item should be regarded as readily biodegradable according to this test.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June 23, 1999 - July 21, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms
Version / remarks:
“Testing Methods for New Chemical Substances” (July 13, 1974, Kanpogyo No.5,
Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615,
Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku
No. 392, Basic Industries Bureau, Ministry of International Trade and Industry,
Japan)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
- Only two vessels were used for test suspensions (test substance with activated sludge in mineral medium) although three vessels are required for 301C.
- There was no vessel of an abiotic control (test substance in water).
Deviations:
yes
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
Activated sludge purchased from Chemicals Evaluation and Research Institute, Japan.
Sludge:
City sewage: Return sludge from sewage plants were collected.
River, lake and sea: Surface water and surface soil which are in contact with the atmosphere were collected.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: % biodegradation by BOD
Reference substance:
aniline
Test performance:
No adverse effects on the reliability of this test were noted.
Key result
Parameter:
other: % biodegradation by BOD
Value:
50
Sampling time:
28 d
Key result
Parameter:
other: % biodegradation by GC
Value:
100
Sampling time:
28 d
Details on results:
The average percentage biodegradation by BOD and GC were 50% and
100%, respectively. In addition, growth of the sludge was observed in the vessels of #8 and 9. However, the average percentage biodegradation by BOD did not meet the criteria for readily biodegradable in the vessels #8 and 9.
From the above result, it was concluded that test material was biodegraded by the activated sludge and has a potential of inherent biodegradability.
Results with reference substance:
Percentage biodegradation of aniline calculated by the BOD values were 43% and 55% after 7 and 28 days, respectively. It was concluded that this test conditions were valid.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is not readily biodegradable (possibly inherent biodegradable).
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
June 23, 1999 - July 21, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structures of the target and source substances are identical and differ only with respect to the ratio of enantiomers where the target substance is a single pure L-isomer and the source substance is an equimolar mixture of L and D isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellyl nitrile, is a mono-constituent substance (EC No. 695-909-8, CAS no. 35931-93-2).
The source substance, DL-Citronellyl nitrile, is a mono-constituent substance (EC No. 257-288-8, CAS no. 51566-62-2).
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the only difference between target and source molecules is the enantiomeric ratio. In a non-chiral environment the target and source chemicals will have identical properties but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). Therefore, as a precaution for the developmental toxicity endpoint it is suggested that the NOAEL 250 mg/kg bw/day for L-Citronellyl nitrile is used instead of 500 mg/kg bw/day, as it is not known which form is more potent in vivo. All other endpoints are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms
Version / remarks:
“Testing Methods for New Chemical Substances” (July 13, 1974, Kanpogyo No.5,
Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615,
Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku
No. 392, Basic Industries Bureau, Ministry of International Trade and Industry,
Japan)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
- Only two vessels were used for test suspensions (test substance with activated sludge in mineral medium) although three vessels are required for 301C.
- There was no vessel of an abiotic control (test substance in water).
Deviations:
yes
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
Activated sludge purchased from Chemicals Evaluation and Research Institute, Japan.
Sludge:
City sewage: Return sludge from sewage plants were collected.
River, lake and sea: Surface water and surface soil which are in contact with the atmosphere were collected.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: % biodegradation by BOD
Reference substance:
aniline
Test performance:
No adverse effects on the reliability of this test were noted.
Key result
Parameter:
other: % biodegradation by BOD
Value:
50
Sampling time:
28 d
Key result
Parameter:
other: % biodegradation by GC
Value:
100
Sampling time:
28 d
Details on results:
The average percentage biodegradation by BOD and GC were 50% and
100%, respectively. In addition, growth of the sludge was observed in the vessels of #8 and 9. However, the average percentage biodegradation by BOD did not meet the criteria for readily biodegradable in the vessels #8 and 9.
From the above result, it was concluded that test material was biodegraded by the activated sludge and has a potential of inherent biodegradability.
Results with reference substance:
Percentage biodegradation of aniline calculated by the BOD values were 43% and 55% after 7 and 28 days, respectively. It was concluded that this test conditions were valid.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is not readily biodegradable (possibly inherent biodegradable).
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14.05.1997 - 16.06.1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structures of the target and source substances are identical and differ only with respect to the ratio of enantiomers where the target substance is a single pure L-isomer and the source substance is an equimolar mixture of L and D isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellyl nitrile, is a mono-constituent substance (EC No. 695-909-8, CAS no. 35931-93-2).
The source substance, DL-Citronellyl nitrile, is a mono-constituent substance (EC No. 257-288-8, CAS no. 51566-62-2).
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the only difference between target and source molecules is the enantiomeric ratio. In a non-chiral environment the target and source chemicals will have identical properties but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). Therefore, as a precaution for the developmental toxicity endpoint it is suggested that the NOAEL 250 mg/kg bw/day for L-Citronellyl nitrile is used instead of 500 mg/kg bw/day, as it is not known which form is more potent in vivo. All other endpoints are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The sludge is collected, in the mineral medium and kept aerobic until being used on the same day.
Dry weight of suspended solids: 6.136 g/L
To obtain a concentration of 30 mgll (dry weight) in a 250 ml flask, 1.22 mL of sludge is needed (inoculum).
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
nominal concentration
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Solution A+B+C+D (see attachment)
- Test temperature: 22°C
- pH: 7.4 ± 0.2
- pH adjusted: yes

TEST SYSTEM
- Culturing apparatus: flasks
- Number of culture flasks/concentration: 2
- Measuring equipment: respirometer
Reference substance:
benzoic acid, sodium salt
Test performance:
- Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.
- At the end of the test period (normally 28 days), the pH of each flask is measured again.
Key result
Parameter:
% degradation (O2 consumption)
Value:
69
Sampling time:
28 d
Details on results:
See attachment.
Key result
Parameter:
ThOD
Value:
2.86 other: mg O2/mg
Results with reference substance:
Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified and the test is considered as valid.

The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test item on the

micro-organisms at the test concentration. On the contrary, biodegradation of the test substance starts much earlier (day 4) in presence of sodium benzoate.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance undergoes on an average 69 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 17-20 after a long lag phase (adaptation of the inoculum) and reaches a mean value of 68 % at the end of the 10-day window (days 17 to 27).
Thus, the test item should be regarded as readily biodegradable according to this test.

Description of key information

This endpoint was fulfilled using read across from 3,7-dimethyloct-6-enenitrile (EC 257-288-8 / CAS 51566-62-2), for which the following results were obtained.

The biodegradation in water was assessed according to test guideline OECD 301 F in the GIVAUDAN-ROURE SA study. The test substance undergoes on an average 69 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 17-20 after a long lag phase (adaptation of the inoculum) and reaches a mean value of 68 % at the end of the 10-day window (days 17 to 27). Thus, the test item should be regarded as readily biodegradable according to this test.

The biodegradation in water was also assessed according to test guideline “Testing Methods for New Chemical Substances” (Ministry of International Trade and Industry, Japan), equivalent or similar to OECD 301 C in the TAKASAGO INTERNATIONAL CORPORATION study. The average percentage biodegradation by BOD and GC were 50% and 100%, respectively. In addition, growth of the sludge was observed in the vessels of #8 and 9. However, the average percentage biodegradation by BOD did not meet the criteria for readily biodegradable in the vessels #8 and 9. The test substance is not readily biodegradable (possibly inherent biodegradable) according to this test.

Considering the above results, the substance was found to be readily biodegradable in the first test. In the second test the substance did not meet the criteria for readily biodegradable. However, the substance did show 50 % biodegradation by BOD after 28 days and has a potential of inherent biodegradability. Therefore, overall the substance can be considered as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

[Type of water: freshwater]