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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-03-2015 to 19-06-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes
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): On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake, and inland sea; return sludge from sewage plants; with sampling period: January 2015 to February 2015). Activated sludge, was prepared in laboratory according to Japan guidelines. Activated sludge, which was cultivated for 19.5 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water and the pH of the solution was adjusted to 7.0 ±1.0.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week after cultivation (19.5 hours typically).
- Preparation of inoculum for exposure: See above.
- Pretreatment: See source of inoculum/activated sludge, above.
- Concentration of sludge: The sludge was diluted in the bottles to 30 mg SS/L.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
The percentage biodegradation by DOC was not calculated because the test item can not be dissolved in water at the test concentration (100 mg/L or more).
Parameter followed for biodegradation estimation:
other: Percentage biodegradation of test item by GC
Details on study design:
TEST CONDITIONS
- Composition of medium: On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake, and inland sea; return sludge from sewage plants; with sampling period: January 2015 to February 2015). Activated sludge, was prepared in laboratory according to Japan guidelines. Activated sludge, which was cultivated for 19.5 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water and the pH of the solution was adjusted to 7.0 ±1.0. Additive amount of activated sludge into the test vessel was 2.60 mL on the basis of the concentration of suspended solid. The basal culture medium (3 L) was prepared at the same proportion as the following method; purified water (Japanese Pharmacopoeia) was added to each 3 mL aliquot of solutions A, B, C and D, which are described in Japan guideline/standard: JIS K 0102-2008 Section 21, in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.
- Additional substrate: Not applicable.
- Solubilising agent (type and concentration if used): Not applicable.
- Test temperature: 25 ±1 °C
- pH: Blank (water + test item): 6.6 ; Sludge + test item: 7.1 – 7.2 ; control blank: 7.3
- pH adjusted: No.
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: 30 mg SS/L.
- Continuous darkness: Yes.
- Other: Not applicable.

TEST SYSTEM
- Culturing apparatus: Glass vessel (ca. 300 mL).
- Number of culture flasks/concentration: Triplicate.
- Method used to create aerobic conditions: Not applicable.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: See previous sections.
- Test performed in closed vessels due to significant volatility of test substance: Not applicable.
- Test performed in open system: Not applicable.
- Details of trap for CO2 and volatile organics if used: Not applicable.
- Other: Not applicable.

SAMPLING
- Sampling frequency: BOD (oxygen consumption) was monitored continuously ; minimum end of incubation for DOC and/or transformation products analysis.
- Sampling method: See above.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not applicable.
- Other: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: Not applicable.
- Toxicity control: Yes.
- Other: Reference item control (Analine)

STATISTICAL METHODS: Not applicable.
Reference substance:
aniline
Remarks:
100 mg/L
Test performance:
1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. Therefore, the test is considered valid (Actual: 3% in BOD)
2. The BOD of the inoculated blank control was 24.8 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for test item vessels)
4. Aniline attained 99% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
5. The BOD of the blank control was 11 mg/L which was < 60 mg/L required.

All validity criteria were considered to be met.
Parameter:
% degradation (O2 consumption)
Remarks:
BOD ; mean
Value:
41
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Remarks:
GC ; mean
Value:
100
Sampling time:
28 d
Details on results:
The test item was degraded based on BOD: 41% at day 28.
The test item was degraded based on GC: 100% at day 28.
The degradation products was determined by GC-MS/LC-MS: 5 detection products were found by GC-MS and ranged from 37% to 1% at day 28.
Identification of the degradants is provided in the 'attached background material'.
Results with reference substance:
Aniline attained 99% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The mean biodegradation in triplicate was 41 % at day 28 by BOD. The mean biodegradation by direct analysis by GC was 100% at day 28. The degradation products were identified and quantified by GC-MS.
Executive summary:

The ready biodegradability test was carried out according to Japanese guidelines and OECD TG 301C guideline under GLP. The test item, at a concentration of 100 mg/L was exposed to mixed activated sewage sludge micro-organisms obtained from on-site sludge sampling carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake, and inland sea; return sludge from sewage plants; with sampling period: January 2015 to February 2015). Activated sludge, was prepared in laboratory according to Japan guidelines. The test item was exposed in culture medium in sealed culture vessels in the dark at 25°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg SS/L at exposure volume of 300 mL. The degradation of the test item was assessed by the measurement of oxygen consumption (BOD) on days 0 through 28 using a closed system oxygen consumption apparatus. DOC was determined quantitatively by TOC analyser (Sievers 900), where applicable. The test item (and degradants) was determined directly quantitatively by Gas Chromatography (GC-MS) and/or qualitatively by LC-MS. Control solutions with inoculum and the reference substance, aniline, together with blank controls were used for validation purposes. In the test inoculum blank the oxygen uptake was 11 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.2 in the test item systems and 6.6 in the test item blank and 7.3 in the blank control system. The repeatability validity criterion of not more than 20% difference between replicates was fulfilled. Therefore, the test is considered valid. The test system met the validation criteria of the guideline. Aniline attained 99% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for triplicate test flasks at 28 days for the test item was 41.0% (minimum 40 and maximum 43%) by BOD. The mean biodegradation by direct analysis by GC was 100% at day 28. The degradation products were identified and quantified by GC-MS. Under the conditions of the study, test item is not considered as readily biodegradable.

Description of key information

Biodegradation: not readily biodegradable ; mean biodegradation 41.0 % BOD and GC 100% (28 -days); transformation products were identified, OECD TG 301C, 2015

Key value for chemical safety assessment

Type of water:
freshwater

Additional information

Key study : OECD TG 301C, 2015 : The ready biodegradability test was carried out according to Japanese guidelines and OECD TG 301C guideline under GLP. The test item, at a concentration of 100 mg/L was exposed to mixed activated sewage sludge micro-organisms obtained from on-site sludge sampling carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake, and inland sea; return sludge from sewage plants; with sampling period: January 2015 to February 2015). Activated sludge, was prepared in laboratory according to Japan guidelines. The test item was exposed in culture medium in sealed culture vessels in the dark at 25°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg SS/L at exposure volume of 300 mL. The degradation of the test item was assessed by the measurement of oxygen consumption (BOD) on days 0 through 28 using a closed system oxygen consumption apparatus. DOC was determined quantitatively by TOC analyser (Sievers 900), where applicable. The test item (and degradants) was determined directly quantitatively by Gas Chromatography (GC-MS) and/or qualitatively by LC-MS. Control solutions with inoculum and the reference substance, aniline, together with blank controls were used for validation purposes. In the test inoculum blank the oxygen uptake was 11 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.2 in the test item systems and 6.6 in the test item blank and 7.3 in the blank control system. The repeatability validity criterion of not more than 20% difference between replicates was fulfilled. Therefore, the test is considered valid. The test system met the validation criteria of the guideline. Aniline attained 99% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for triplicate test flasks at 28 days for the test item was 41.0% (minimum 40 and maximum 43%) by BOD. The mean biodegradation by direct analysis by GC was 100% at day 28. The degradation products were identified and quantified by GC-MS. Under the conditions of the study, test item is not considered as readily biodegradable.

 

The applicant addressed the n-Octanol/Water partition coefficient of the identified degradants observed in the current OECD TG 301C MITI test using an OECD TG 117 screening test. None of the degradants have Log Kow > 3.4.

Details of which are attached by the applicant.