Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-08 to 2014-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Test temperature fell below 22 ± 2°C on three occasions. As this was a short period and the temperature consistent throughout the rest of the study period it was not considered to have an adverse effect.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
see above
Principles of method if other than guideline:
Following the recommendations of ISO 1995 for organic substances with low water solubility, the test item was dissolved in an auxillary solvent prior to being absorbed onto filter paper and subsequent dispersal in the test media using high shear mixing to break up the filter paper. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: clear colourless liquid
- Storage condition of test material: room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- See read-across justification attached.

Study design

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): obtained on 7 April 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Preparation of inoculum for exposure: The sample waswashed twice by settlement and resuspension in mineral medium to remove excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection.
- Suspended solids concentration was equal to 2.9 g/L prior to use.
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper
Duration of test (contact time):
>= 28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Prepared to OECD Guideline
- Solubilising agent (type and concentration if used): The test item was dissolved in an auxillary solvent prior to adsorption onto filter paper and then added to the test medium using high shear mixing.
An amount of test item (792 mg) was dissolved in 10 mL of acetone to give a 792 mg/10 mL solvent stock solution. An aliquot (500 µL) of this solvent stock solution was dispersed onto filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed into approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to innoculated mineral medium. The volume was then adjusted to 3 litres to give a final concentration of 13.2 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
- Test temperature: 2°1C
- pH: 7.5 - 7.6
- pH adjusted: no
- Aeration of dilution water: No
- Suspended solids concentration: final: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2 free air bubbled through solution at a rate of 30 to 100 mL/min
- Measuring equipment: IC analysis using a Tekmar=Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser and a Shimadzu TOC-VCPH analyser.
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics if used: Two 500 mL Dreschel bottles containing 350 mL of 0.05M NaOH. CO2 absorbing solutions prepared using purified de-gassed water.

SAMPLING
- Sampling frequency: Days 0, 2, 6, 8, 10, 14, 21, 28 and 29.
- Sampling method: 2mL removed from first CO2 absorber vessel
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate vessels consistingof inoculated mineral medium plus a filter paper
- Abiotic sterile control: No
- Toxicity control: Test item plus sodium benzoate to give a final concentration of 20 mg carbon/L
- Other: Sodium benzoate in duplicate plus filter paper to give a final concentration of 10 mg carbon/L
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Not applicable. The test item is an unstable UVCB substance in aqueous conditions and hydroyses rapidly (less than 30 minutes). The water solubility of the test item is therefore equivalent to the water solubility of the two main degradation products boric acid and 2-propyl heptan-1-ol. Both degradation products have been registered under REACH. Boric acid is an inorganic substance with high water solubility and will not undergo biodegradation. 2-propyl heptan-1-ol is a poorly soluble substance (> 100 mg/L) and is readily biodegradable with over 90% degradation in 28-days.
Test performance:
The total CO2 evolution on the inoculum control vessels on Day 28 was 33.38 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given inthe oECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation croterion given in the OECD Test Guidelines.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
40
Sampling time:
8 d
Parameter:
% degradation (CO2 evolution)
Value:
50
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
66
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
74
Sampling time:
29 d
Remarks on result:
other: Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2
Details on results:
The results of the inorganic carbon analysis of samples from the first absorber vessels om Day 29 showed an increase in all replicate vessels with the exception of inoculum control R2 and procedure control R2.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over into the second absorber vessels occurred.
The test item attained 74% biodegradation after 28 days. As the test material is a UVCB the 10 -day window validation criterion are not applicable. Therefore, the test material can be considered to be readily biodegradable.The organic degradation product derived from hydrolysis of the test item is readily biodegradable.
The toxicity control attained 69% biodegradation after 14 days and 77% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatement micro-organisms in the test.
Sodium benzoate attained 82% biodegradation after 14 days and 87% biodegradation after 28 days confirming the suitability of the inoculum and test conditions.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 82% biodegradation after 14 days and 87% after 28 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 74% biodegradation after 28 days. The test item is readily biodegradable
Executive summary:

Guidance

OECD Guideline No. 301B and EC Method C.4-C : CO2Evolution test

Method

The analogue test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of ISO 1995, the test item was dissolved in an auxiliary solvent prior to being absorbed onto filter paper and subsequent dispersal in the test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 74% biodegradation after 28 days. As the test material is a UVCB the 10 -day window validation criterion are not applicable. Therefore, the test material can be considered to be readily biodegradable.The organic degradation product derived from hydrolysis of the test item is readily biodegradable.

The test item is an unstable UVCB substance in aqueous conditions and hydroyses rapidly (less than 30 minutes). The water solubility of the test item is therefore equivalent to the water solubility of the two main degradation products boric acid and 2-propyl heptan-1-ol. Both degradation products have been registered under REACH. Boric acid is an inorganic substance with high water solubility and will not undergo biodegradation. 2-propyl heptan-1-ol is a poorly soluble substance (> 100 mg/L) and is readily biodegradable with over 90% degradation in 28-days.

The toxicity control reached 69% biodegradation after 14 days and 77% biodegradation after 28 days. Sodium benzoate attained 82% biodegradation after 14 days and 87% after 28 days.

Conclusion

The analogue test item attained 74% biodegradation after 28 days. The test item is readily biodegradable