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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:
19.10.2017-28.12.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yasho Industries Pvt. Ltd. / 40003010617
- Expiration date of the lot/batch: 2019 February
- Purity test date: 2017 March


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: test item formed an emulsion in mineral medium at 220 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not observed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified
- Preliminary purification step (if any): no
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, adapted
Details on inoculum:
- Source of inoculum (e.g. location, sampling depth, contamination history, procedure): Sewage Treatment Plant Bengaluru - 560058 India
- Storage conditions: at room temperature, aerated
- Storage length: 5 days
- Preparation of inoculum for exposure: The effluent was allowed to settle for one hour, decanted and the decanted effluent was used in the test
- Initial cell/biomass concentration: 7.0 x 10^7 CFU/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
>= 10 - <= 20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mixture of 24 mL of stock solution A with 1920 mL of Milli-Q water, then 2.4 mL each of stock solutions B, C and D was added and finally the volume was made up to 2400 mL using Milli-Q water (See tables)
- Test temperature: 21-24 C
- pH: approx. 7.8
- pH adjusted: no
- Aeration of dilution water: with CO2 free air
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Conical flask 5 L - each fitted with an aeration tube reaching nearly to the bottom of the flask and an outlet
- Number of culture flasks/concentration: 2 / 10-20 mg/L
- Method used to create aerobic conditions: The flasks were aerated with CO2 free air at 46 to 50 mL/minute
- Test performed in closed vessels due to significant volatility of test substance: no
- Details of trap for CO2 and volatile organics if used:

SAMPLING
- Sampling frequency: Days 2, 4, 7, 9, 11, 14, 18, 21, 25, 29
- Sampling method: the CO2 was trapped in barium hydroxide and was measured by titration of the residual hydroxide or as inorganic carbon

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flasks
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 10.12
St. dev.:
0.608
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 68.03
St. dev.:
5.721
Sampling time:
11 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 92.11
St. dev.:
2.729
Sampling time:
29 d

Table 1: Carbon dioxide Production

Day CO2 Production (mg) Cumulative CO2 produced (mg)
Test - Blank Mean
Test Flasks Blank
Flask 1 Flask 2 Flask 5 Flask 6 Flask 3 Flask 4 Mean Flask 1  Flask 2 Flask 5  Flask 6
Day 2 30.14 31.68 36.63 31.24 12.54 11.88 12.21 17.93 19.47 24.42 19.03
Day 4 39.49 34.54 41.25 31.46 12.98 14.19 13.59 43.83 40.42 52.08 36.9
Day 7 41.03 43.56 44.55 38.61 9.68 12.65 11.17 73.69 72.81 85.46 64.34
Day 9 37.18 33.33 41.14 33.55 13.86 11.77 12.82 98.05 93.32 113.78 85.07
Day 11 29.48 48.73 17.16 28.93 13.53 11.88 12.71 114.82 129.34 118.23 101.29
Day 14 25.19 10.89 13.75 17.38 9.9 11.44 10.67 129.34 129.56 121.31 108
Day 18 30.58 31.68 34.1 19.91 21.89 19.36 20.63 139.29 140.61 134.78 107.28
Day 21 13.64 10.01 10.34 41.14 0.88 1.98 1.43 151.5 149.19 143.69 146.99
Day 25 24.09 14.08 20.24 12.54 7.48 0.88 4.18 171.41 159.09 159.75 155.35
Day 29 5.17 10.56 3.19 5.83 10.67 4.95 7.81 168.77 161.84 155.13 153.37

Total CO2 produced in the inoculum blank during the test = 107.22 mg/L

= 35.74 mg/L

Note: Flask 1 and 2: Contain test item (methyl eugenol) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

Table 2: ph of Test Solutions

Flask No. pH at the end of the test (Day 28)
Flask 1 7.84
Flask 2 7.83
Flask 3 7.81
Flask 4 7.85
Flask 5 7.82
Flask 6 7.83

Note: Flask 1 and 2: Contain test item (methyl eugenol) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

Table 3: Degradation of Test Item and Reference Item

Day Test Item Refernce Item Test Item + Reference Item
Flask 1 Flask 2 Flask 3
Day 2 9.99 10.85 10.42 14.69 10.94
Day 4 24.42 22.52 23.47 31.33 21.21
Day 7 41.06 40.57 40.82 51.4 36.99
Day 9 54.64 52 53.32 68.44 48.9
Day 11 63.98 72.07 68.03 71.12 58.23
Day 14 72.07 72.19 72.13 72.97 62.08
Day 18 77.61 78.35 77.98 81.07 61.67
Day 21 84.42 83.13 83.78 86.43 84.5
Day 25 95.51 88.65 92.08 96.09 89.3
Day 29 94.04 90.18 92.11 93.31 88.16

% degradation = (mg CO2 produced / (mg TOC added in test x 3.67)) x 100

Note: Flask 1 and 2: Contain test item (methyl eugenol) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (sodium benzoate) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results, the test item was concluded as readily biodegradable as 68.03% mean degradation was achieved in between Days 2 and 11, crossing 60% pass level in 10-day window period after reaching 10% of ThCO2 production and within the 28-d period of the test.
Executive summary:

The ready biodegradability of Methyl Eugenol was tested using the CO2 Evolution Test. The test item was added to two test vessels at the concentration of 66 mg/L of mineral medium (equivalent to 16.3 mg of Total Organic Carbon/L). Two control treatments containing only the inoculum, one reference item treatment and one toxicity control treatment containing the test item and the reference item were also tested. All the treatments were added with equal volume of inoculum which was collected from the secondary effluent treatment plant receiving predominantly domestic sewage.

Treatment mixtures were aerated for 29 days with carbon dioxide (CO2) free air. The CO2 released was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on Days 2, 4, 7, 9, 11, 14, 18, 21, 25 and 29 after the initiation of the test.

The mean per cent degradation of test item was 92.11% at the end of the test while, the percent degradation of reference item was 93.31% and the toxicity control was 88.16% at the end of the test. It was observed that the mean degradation of test item reached 68.03% on Day 11 after reaching 10.12% on Day 2. Therefore, the degradation of test item, Methyl Eugenol reached more than 60% pass level in 10 -day window period after reaching 10% of ThCO2 production and within the 28 -d period of the test.

The test fulfilled all the validatity criteria.

Based on the results, the test item was concluded as readily biodegradable as 68.03% mean degradation was achieved in between Day 2 and Day 11, crossing 60% pass level in 10 day window period after reaching 10% of ThCO2 production and within the 28 day period of the test.

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:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
1. SOFTWARE
Danish QSAR database
2. MODEL (incl. version number)
BIOWIN 5 and BIoWIN 6
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
c1(OC)c(OC)cc(CC=C)cc1
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint: not specified
- Unambiguous algorithm: not specified
- Defined domain of applicability: not specified
- Appropriate measures of goodness-of-fit and robustness and predictivity: not specified
- Mechanistic interpretation: not specified

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: not specified
- Structural and mechanistic domains: not specified
- Similarity with analogues in the training set: not specified
- Other considerations (as appropriate): not specified

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
The prediction supports the findings of the test results, that the substnace is readily biodegradable.
Qualifier:
no guideline available
Principles of method if other than guideline:
- Software tool(s) used including version: Danish QSAR database
- Model(s) used: BIOWIN 5 and BIOWIN 6
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
0.561
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
Biowin 5
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
0.595
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
Biowin 6
Interpretation of results:
readily biodegradable
Conclusions:
According to the QSAR predictions of the Danish QSAR database the substance considered readily biodegradable.
Executive summary:

According to the QSAR predictions of the Danish QSAR database the substance considered readily biodegradable.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information