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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 June 2003 - 8 September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate included in report

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class no. 3.1.1.3)
Molecular formula:
Not applicable, see remarks.
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class no. 3.1.1.3)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPW 21180
- Expiration date of the lot/batch: 19 March 2012
- Stability under test conditions: The test material and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: - 20⁰C

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Oakley sewage treatment works ( UK).
- Preparation of inoculum for exposure: Aliquots (25 mL) of homogenised sample were filtered through dried and pre-weighed Whatman GFC filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.

Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral Salts Medium: 10 mL Stock solution 1 (potassium dihydrogen phosphate: 8.5g/L; di-potassium hydrogen phosphate: 21.75 g/L; di-sodium monohydrogen phosphate dihydrate: 33.4 g/L; ammonium chloride: 0.5 g/L) was mixed with 800 mL dilution water (Tap water softened and treated by reverse osmosisn and then purified) and then adding 1 mL magnesium sulphate heptahydrate (22.5 g/L), 1 mL calcium chloride dihydrate (36.4 g/L) and 1 mL iron(III)chloride hexahydrate (0.25 g/L) for each litre of water required for the test.
- Test temperature: 20.1 - 23.8°C
- pH: 7.6 at the start
- pH adjusted: to 7.4 ± 0.2 with 5M HCl
- Aeration of dilution water: air-saturated
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Number of culture flasks/concentration: 2 control and 2 test substance, 1 reference, and 1 toxicity control
- Method used to create aerobic conditions: Test vessels were continuously flushed for 29 days with treated air.
- Test performed in closed vessels: yes
- Details of trap for CO2 : The air outlet from each vessel was connected to three Dreschel bottles in a series, each containing 0.025N, nominal barium hydroxide (100 mL).
- Other: The residual concentrations of barium hydroxide in the bottle nearest to the test vessels were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphtalein indicator. Following the removal of the first Dreschler bottle in a series, the second was connected to the test vessel and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral Salts Medium
- Toxicity control: Test substance (10 mg C/L) plus reference substance (10 mg C/L) in inoculated mineral medium
- Reference substance: Sodium benzoate in inoculated mineral medium at 10 mg C/L

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
6 d
Parameter:
% degradation (CO2 evolution)
Value:
105
Sampling time:
29 d

BOD5 / COD results

Results with reference substance:
The degradation of sodium benzoate achieved 65% of its TCO2 after 6 days and 88% after 29 days. Also in the presence of the test substance cutinase, PPW 21880, sodium degradation of benzoate achieved 65% of its TCO2 after 6 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Cutinase, PPW 21880 was found to be readily biodegradable in the CO2 evolution test.
Executive summary:

Introduction

The ready biodegradability of Cutinase, PPW 21180 was assessed in the CO2Evolution test (Modified Sturm Test) Procedure C.4-C of the Annex to Directive 93/69/EEC, OECD Procedure 301B and US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances, (OPPTS) Method 835.3110 (m) (adopted January 1998).

 

RESULTS

Mean cumulative CO2production by mixtures containing Cutinase, PPW 21180 was equivalent to 10% of the theoretical value by Day 2 and 62% on Day 6. Substances are considered to be readily biodegradable in this test if CO2production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. Therefore, Cutinase, PPW 21180 can be considered to be readily biodegradable.

 

By the end of the test on Day 29, cumulative CO2evolution was >100% of the theoretical level (replicates 109% and 102%, mean 105%). However, as Cutinase, PPW 21180 had achieved the pass criterion for ready biodegradation within a four-day period, this imprecision in the test system is not considered to have affected the interpretation of this investigation.

CONCLUSION

Cutinase, PPW 21180 can be considered to be readily biodegradable.