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Environmental fate & pathways

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:
20 February - 21 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Version / remarks:
(1999)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.

- Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.5 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant and confirmed by determination of the amount of suspended solids). Before use, the sludge was allowed to settle (30 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
18 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon (TOC)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: varied between 22 and 23°C
- pH:
At t=0 d: 7.6
At t=28 d: 7.6 – 7.9
- pH adjusted:no
- Aeration of test medium: Overnight prior to the start of the test. During the test the medium was aerated and stirred continously.
- Suspended solids concentration: The concentration of suspended solids was 4.5 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (30 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/L of mineral medium.


- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), with CO2 < 1 ppm) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made 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 at least 14 days.


SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 9, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
other: Sodium acetate
Preliminary study:
- Preparation of test solutions: Since Accelerator (PT 25E or PT 25E/2) was easily soluble in water the test media were prepared using a stock solution of 1 g/L in Milli-RO water (tap water purified by reverse osmosis). A weighed amount of 501.6 mg of Accelerator (PT 25E or PT 25E/2) was dissolved in Milli-RO water and made up to 500 ml. Aliquots of 36 ml of the clear and colourless stock solution were added to the test substance bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1.5
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day)
Details on results:
- Theoretical CO2 production:
The ThCO2of Accelerator (PT 25E or PT 25E/2) was calculated to be 2.44 mg CO2/mg.
The ThCO2of sodium acetate was calculated to be 1.07 mg CO2/mg.

- Biodegradation:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Accelerator (PT 25E or PT 25E/2). The biodegradation of the test substance measured in the 2 test bottles at day 29 was 1 and 2 %.
In the toxicity control more than 25% biodegradation occurred within 14 days (35%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.
Results with reference substance:
The positive control substance was biodegraded by at least 60% (74%) within 14 days.

Acceptability of the test

1.    The positive control substance was biodegraded by at least 60% (74%) within 14 days.

2.    The difference of duplicate values for %-degradation of the test substance was always less than 20.

3.    The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (55 mg CO2 per 2 litres of medium, corresponding to 28 mg CO2/L).

4.    The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/L).

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
- Accelerator (PT 25E or PT 25E/2) was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The readily biodegradation of Accelerator (PT 25E or PT 25E/2) under the conditions of the carbon dioxide (CO2) evolution test (modified Sturm test) was investigated according to OECD guideline 301 B and GLP principles. A single test concentration of 12 mg/L (as TOC) was tested during 28 days. Based on the obtained results, Accelerator (PT 25E or PT 25E/2) was not readily biodegradable. The study is considered to be reliable without restrictions.

Description of key information

The readily biodegradation of Accelerator (PT 25E or PT 25E/2) under the conditions of the carbon dioxide

(CO2) evolution test (modified Sturm test) was investigated according to OECD guideline 301 B and GLP

principles. Accelerator (PT 25E or PT 25E/2) was not readily biodegradable. The study is considered to be

reliable without restrictions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The readily biodegradation of Accelerator (PT 25E or PT 25E/2) under the conditions of the carbon dioxide (CO2) evolution test (modified Sturm test) was investigated according to OECD guideline 301 B and GLP principles. A single test concentration of 12 mg/L (as TOC) was tested during 28 days. Based on the obtained results, Accelerator (PT 25E or PT 25E/2) was not readily biodegradable. The study is considered to be reliable without restrictions.