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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:
14 Apr - 13 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 9115
- Substance type: pure active substance
- Physical state: Clear, colorless to yellowish liquid
- Analytical purity: 96%
- Expiration date of the lot/batch: 24 Nov. 2015
- Stability under test conditions: stable
- Storage condition of test material: at room temperature

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions:The freshly obtained sludge was kept under continuous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle 33 min and the supernatant was used at the amount of 10 mL per liter of mineral medium
- Pretreatment: Mineral medium, dilution water, and inoculum (1% of final volume) were added to the test flasks on the day before the test. Flasks were aerated overnight to purge the system of CO2.
- Concentration of sludge: Suspended solids concentration was 4.7 g/L
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
16.5 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 (ca. 20% oxygen, 80% nitrogen, <1 ppm CO2) overnight to purge the media of CO2 before start of test and during test to provide oxygen and to remove CO2.
- Test temperature: 21.6 - 22.4 °C
- pH: pH adjusted to 7.5 at start of test. Positive control and toxicity control had final pH of 7.8. Blank and test substance had final pH 7.5.
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 4.7 mg/L
- Continuous darkness: The test media were excluded from light

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 was sparged through 0.0125M barium hydroxide to remove any CO2 present prior to use to aerate the test system. Synthetic air sparge rate was ca. 30-100 mL/min.
- Measuring equipment: CO2 production determined by titrating remaining barium hydroxide with 0.05 M standardized HCl
- Test performed in open system: yes (CO2-free synthetic air blown continuously through medium)
- 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 mixed (by vortex) 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.
- 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 each bottle. The bottles were aerated overnight to drive off any CO2 present. The final titrations were made on day 29.
- 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
- Positive control: 40 mg/L sodium acetate
- Toxicity control: Contained test substance at ca. 16.5 mg/L and sodium acetate at 40 mg/L plus mineral medium and inoculum (12 mg TOC/L in each substance)

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
7 - 12
Sampling time:
28 d
Remarks on result:
other: values for each of duplicate flasks
Details on results:
The ThCO2 of Procrylat was calculated to be 2.66 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Mean CO2 evolution in blanks was 44 mg/2L (22 mg CO2/L) at the end of the tests, which is within limits of OECD TG301B (<40 mg/L).

CO2 evolution for the reference substance demonstrated 9% biodegradation on Day 3, reaching 60% by Day 10 and 71% by Day 14.

CO2 evolution in test substance bottles was in the same range as inoculum blanks at the first time point. Therefore, the amount of inorganic carbon in the test substance suspension at the start of the experiment was negligible (<5% of total carbon in the test substance suspension)

CO2 evolution in the toxicity control was 36% of ThCO2 on day 14. No inhibition by Procrylat is expected.

% biodegradation vs time plot - see Figure 1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
positive control 71% degraded within 14 d, difference in deg of test substance 5%, total CO2 from blank 22 mg CO2/L, inorganic carbon in test substance <5% of total carbon.
Interpretation of results:
other: not readily biodegradable
Conclusions:
Procrylat was not readily biodegradable in an OECD 301B test (7-12% CO2/ThCO2)
Executive summary:

Procrylat was tested in an OECD301B test, using activated sludge from predominantly domestic sewage as inoculum. Cumulative CO2 release in duplicate flasks was 7% and 12% of theoretical relative to inoculum blanks. The sodium acetate positive control was 71% degraded within 14 days. The toxicity control, containing 12 mg C/L of both test and reference substance, showed 36% CO2/ThCO2 after 14 days. Procrylat is not readily biodegradable, but is not expected to inhibit biodegradation.

The test was done according to internationally accepted guidelines under GLP criteria. This study is considered reliable without restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.