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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11/August/2010 - 9/September/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The work reported was performed carefully, the results reasonable, and the conclusions conservative. Study was conducted in accordance with OECD guidance. Results for test and reference substances met criteria for a valid test result.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Remarks:
At day 14 the reference substance showed > 60% biodegradation; duplicate flasks had less than 20% difference in % biodegradation; the inorganic carbon content of the test substance in mineral medium at test initiation was < 5% of the total carbon content.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 21721
- Physical state: Colorless liquid
- Lot/batch No.: TFEE5- 276/12/09-1
- Expiration date of the lot/batch: 31 December 2012
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature in the dark under nitrogen

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the "Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, municipal sewage treatment plant.
- Storage conditions: Continuous aeration until use.
- Storage length: Not reported ("freshly obtained")
- Preparation of inoculum for exposure: Sludge allowed to settle 35 minutes and liquid decanted for use as inoculum.
- Pretreatment: None
- Concentration of sludge: 10 mL/L mineral medium
- Initial cell/biomass concentration: Not reported.
- Water filtered: Not reported
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
56 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard mineral media as per OECD 301B. Aerated with synthetic air overnight to purge the media of CO2.
- Test temperature: 21.6 - 22.2 deg C
- pH: 7.6 - 8.6
- pH adjusted: No
- CEC (meq/100 g): Not Reported
- Aeration of dilution water: Aerated each test system (without test or reference substance) with synthetic air (CO2 < 1 ppm) overnight prior to test initiation
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported. Test vessels were brown-colored bottles
- Other: Test media aerated and stirred continuously during the test period.

TEST SYSTEM
- Culturing apparatus: 2-liter all-glass brown colored bottles
- Number of culture flasks/concentration: 2 for test suspension; 2 for inoculum blank, 1 for reference substance, 1 for toxicity control.
- Method used to create aerobic conditions: Continuous aeration and stirring. Synthetic air (ca. 20% oxygen and ca. 80% nitrogen) was passed through 0.0125M barium hydroxide to remove any CO2 present prior to use to aerate the test system
- Measuring equipment: CO2 production determined by titrating remaining barium hydroxide with 0.05 M standardized HCl
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: 100 mL of 0.0125 M Ba(OH)2) in each of three bottles connected in series to the exit air line of each exposure flask.
- Other: Test substance was not sufficiently water soluble to allow preparation of an aqueous stock solution. Weighed amounts of test substance and then 10 mL of Milli-RO water were added to weighing bottles. The weighing bottles were vigorously shaken and the resulting suspension was added quantitatively to the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and the microorganisms.

SAMPLING
- Sampling frequency: Every second or third day during the first 10 days and thereafter at least every fifth day until the 28th day for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made 5 times through day 14.
- Sampling method: At each sample time, the CO2 absorber nearest to the test bottle was removed for analysis. Each of the remaining two absorbers was moved one position in the direction of the test bottle and a new absorber was placed at the far end of the series. Phenolphthalein was used as a pH indicator. On the 28th day, the pH of all test solutions was measured and 1 mL of 37% HCl was added to the inoculum blank and test suspension bottles. The bottles were aerated overnight to drive off any CO2 present. The final titrations were made on day 29.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: None.
- Other: The theoretical CO2 production was calculated from the molecular formula.
CONTROL AND BLANK SYSTEM
- Inoculum blank: Contained only mineral media and inoculum
- Abiotic sterile control: None
- Toxicity control: Contained test substance at 56 mg/L and sodium acetate at 40 mg/L plus mineral medium and inoculum
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
N/A
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
28 d
Details on results:
The ThCO2 of TFEE-5 was calculated to be 0.79 mg CO2/mg based on structure
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg based on structure.

- Effect concentrations exceeding solubility of substance in test medium: Yes

% biodegradation vs time plot - see attachment 1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
At day 14 the reference substance showed > 60% biodegradation; duplicate flasks had less than 20% difference in % biodegradation; the inorganic carbon content of the test substance in mineral medium at test initiation was < 5% of the total carbon content.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
TFEE-5 was not readily biodegradable under test conditions
Executive summary:

The ready biodegradability of TFEE-5 was studied in a 28-day CO2 evolution test according to OECD method 301B. Activated sludge from a domestic sewage treatment plant was used as the source of inoculum. Brown glass bottles were filled with oxygen-saturated, CO2-free medium and spiked with one of the following: TFEE-5 at 56 mg/L (12 mg TOC/L), sodium acetate (reference substance) at 40 mg/L (12 mg TOC/L), TFEE-5 plus sodium acetate (toxicity control), or inoculum alone (inoculum blank). Evolved CO2 was titrated every second or third day during the first 10 days and thereafter at least every fifth day until the 28th day for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made 5 times through day 14. The 28-day biodegradation of TFEE-5 was found to be 3% by CO2 evolution. In the toxicity control more than 25% biodegradation occurred within 14 days (33%) therefore the test substance did not inhibit microbial activity. The reference substance sodium acetate showed 63% biodegradation at 14 days demonstrating the viability of the inoculum. Based on these data, TFEE-5 was found to not be readily biodegradable under the conditions of the CO2 evolution test.

This study is classified as acceptable and satisfies the guideline requirements for test method OECD301B, ready biodegradability: CO2 evolution test.

Results Synopsis

Test Organism Age: less than one day

Test Type: Open system

Percent degradability: 3%                     95% C.I.: not applicable