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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:
June 2016 - August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: hydrogenious 005
- Expiration date of the lot/batch: >3 years, unlimited shelf life
- Purity: > 95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature
- Stability under test conditions: stable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Cambridge Sewage Treatment Works, Cowley Road, collected on 05 July 2016
- Pretreatment of sludge: Sieved to 850μm to remove coarse particulates, settled and centrifuged at ~ 4000rpm for ~ 5-10 minutes. The supernatant discarded, sludge re-suspended in mineral media and centrifuged at ~ 4000rpm for ~ 5-10 minutes. This stage repeated once more then the supernatant discarded again and homogenised thoroughly by mechanical stirring (spoon). Dry sludge solid detemined on the pellet produced.
- Concentration of sludge: 7.31% dry sludge solids
- Initial cell/biomass concentration: 0.030 g/l dry sludge solids in test
Duration of test (contact time):
28 d
Initial conc.:
19.99 mg/L
Based on:
TOC
Remarks:
Test vessel 1
Initial conc.:
20.05 mg/L
Based on:
TOC
Remarks:
Test vessel 2
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium acc. to guideline
- Test temperature: 22 ± 2 °C
- Suspended solids concentration: 0.030 g/l
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 1 wash bottle, filled with soda lime to remove CO2 from the atmosheric air pumped through the system, followed by the test vessel, followed by 2 carbon dioxide traps
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: atmospheric air pumped into the test system
- Measuring equipment: Dissolved Inorganic Carbon (DIC) measured using a Tekmar-Dohrmann Phoenix 8000 (UV-Persulfate Analyser)
- Details of trap for CO2: 200 ml of 0.05 M sodium hydroxid solution

SAMPLING
- Sampling frequency: at days 2, 5, 7, 9, 14, 19, 23, 28, and 29

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: none
- Toxicity control: 1
Reference substance:
acetic acid, sodium salt
Remarks:
CAS No.: 127-09-3, supplied by Fisher scientific, Lot/batch number: 1280233, expiry date: April 2017, composition and purity: ≥99%
Test performance:
Conical flasks of nominal volume 2000ml were filled with 1500ml of inoculated mineral medium. The blanks, reference and test bottles were set up in duplicate, with a single replicate for the toxicity control. Test and reference materials were added to appropriate bottles to a final concentration of 20mg Carbon/l respectively. Atmospheric air was pumped into the test system and scrubbed clean of carbon dioxide by passing over soda lime. The air continued into the test vessel where it collects any evolved carbon dioxide before moving into the carbon dioxide traps, each containing 200ml of 0.05M sodium hydroxide solution. The test solutions were stirred for the duration of the study. The carbon dioxide evolved from each bioreactor flask was trapped in 200ml aliquots of 0.05M sodium hydroxide which was sacrificially sampled at specific time points. On day 28, 2ml of 50% (v/v) hydrochloric acid was added to each bioreactor, which were then aerated overnight, to drive off the remaining carbon dioxide. One last analysis of evolved carbon dioxide was made on day 29. The toxicity control was prepared by adding to the flask the same quantities of carbon as used in each of the respective test and reference material flasks (20mg/l of each). The degradation of the reference material in the toxicity control was determined by using the average test sample value as the “blank” at each time point.
The concentration of carbon dioxide was determined as inorganic carbon (mg/l) and converted to mg carbon in 200ml of sodium hydroxide (concentration x 200/1000). The cumulative quantities of carbon (adjusted for the average cumulative blank) were determined for each of the blank, reference, test and toxicity control vessels. The percentage degradation was calculated for all flasks at each of the time points.
degradation (%) = (mg inorganic carbon (test/reference/toxicity control) – mg average blank) x 100 / mg carbon (test/reference/toxicity control)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d

Table: Percentage degradation     

Time [days] Test substance Reference
(sodium acetate)
Toxicity control
vessel 1 vessel 2 mean mean
0 0 0 0 0 0
2 -1 0 -1 29 8
5 -3 -1 -2 48 21
7 -3 -1 -2 54 25
9 -4 -2 -3 58 27
14 -4 -2 -3 64 30
19 -1 -4 -2 64 29
23 -2 -4 -3 66 30
28 -2 -4 -3 67 31
29 -1 -4 -3 70 33
29 1 -5 -2 81 40
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance dibenzylbenzene, ar-methyl derivative, hydrogenated is not readily biodegradable under the conditions of this test.
Executive summary:

The test substance dibenzylbenzene, ar-methyl derivative, hydrogenated was determined in a Ready Biodegradability – CO2 Evolution test according to OECD Guideline 301B in compliance with GLP. Dibenzylbenzene, ar-methyl derivative, hydrogenated failed to meet the requirements for a pass in this test (≥60% degradation relative to the ThCO2 value) with a maximum of -1% (this dropped to -3% during the study). The inoculum blank should not normally produce more than 40mg/l CO2 (10.9mg/l C) for the test to be valid. A value of 26.9mg/l CO2 (7.3mg/l C) was recorded. The guideline requires that the reference material degrades by at least 60% of its ThCO2 value, a maximum value of 81% degradation of the sodium acetate was achieved on day 29.  A toxicity control containing dibenzylbenzene, ar-methyl derivative, hydrogenated and the reference material (both at 20mg/l) was run in parallel. Inspection of the data generated indicates that, whilst dibenzylbenzene, ar-methyl derivative, hydrogenated was not found to be degradable, it was also not inhibitory to the micro-organisms responsible for the degradation of the reference material.

The final conclusion of the study is therefore that dibenzylbenzene, ar-methyl derivative, hydrogenated is not readily biodegradable under the conditions of this test.

Description of key information

The test substance dibenzylbenzene, ar-methyl derivative, hydrogenated was determined in a Ready Biodegradability – CO2 Evolution test according to OECD Guideline 301B in compliance with GLP. Dibenzylbenzene, ar-methyl derivative, hydrogenated failed to meet the requirements for a pass in this test (≥60% degradation relative to the ThCO2 value) with a maximum of -1% (this dropped to -3% during the study). The inoculum blank should not normally produce more than 40mg/l CO2 (10.9mg/l C) for the test to be valid. A value of 26.9mg/l CO2 (7.3mg/l C) was recorded. The guideline requires that the reference material degrades by at least 60% of its ThCO2 value, a maximum value of 81% degradation of the sodium acetate was achieved on day 29.  A toxicity control containing dibenzylbenzene, ar-methyl derivative, hydrogenated and the reference material (both at 20mg/l) was run in parallel. Inspection of the data generated indicates that, whilst dibenzylbenzene, ar-methyl derivative, hydrogenated was not found to be degradable, it was also not inhibitory to the micro-organisms responsible for the degradation of the reference material. The final conclusion of the study is therefore that dibenzylbenzene, ar-methyl derivative, hydrogenated is not readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information