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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:
From 11 April, 2012 to 6 June, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.1 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (45 min) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
ca. 28 d
Initial conc.:
18 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 L 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 L, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 L.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 L.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 L.

- Test temperature: between 21.8 and 22.3°C.
- pH:
At t=0 d: 7.4 - 7.6
At t=28 d: 7.5 – 7.9
- pH adjusted: only before the start of the test from 7.7-7.8 to 7.4-7.6
- Aeration of dilution water: Not before the test, the test is aerated continuously
- Suspended solids concentration: The concentration of suspended solids was 3.1 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (45 min) 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 L 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%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Test performed in open system: Yes
- 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 d, and thereafter at least every fifth day until the 28th d, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 d.


SAMPLING
- Sampling frequency: Titration were made on Days 3, 6, 8, 10, 14, 17, 22, 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:
acetic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 0
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 2
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day)
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of DPHA. In the toxicity control more than 25% biodegradation occurred within 14 days (41%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The positive control substance was biodegraded by at least 60% (81%) within 14 d.

Theoretical CO2 production: The TOC of DPHA was determined to be 66%. ThCO2 was calculated to be 2.42 mg CO2/mg.The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Tables: Refer to the attached pdf under 'attached background material'.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered as not readily biodegradable (Desmares-Koopmans, 2012).
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, DPHA, using the CO2 evolution (modified Sturm) method according to OECD Guideline 301B, in compliance with GLP. In addition, the procedures were designed to meet EU Method C.4-C, ISO International Standard 9439 (1999) and ISO Standard 10634 (1995). The test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. Since the organic carbon content could not be calculated, a sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC) content. The TOC content of DPHA was determined to be 66%. The test substance was tested in duplicate at 18 mg/l, corresponding to 12 mg TOC/l. Based on the TOC content the ThCO2 of DPHA was calculated to be 2.42 mg CO2/mg. The study consisted of six bottles:(a) 2 inoculum blanks (no test substance) (b) 2 test bottles (DPHA) (c) 1 positive control (sodium acetate) (d) 1 toxicity control (DPHA plus sodium acetate). Weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, approximately 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex), the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29th day). The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of DPHA. In the toxicity control, DPHA was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the test substance was considered as not readily biodegradable (Desmares-Koopmans, 2012).

Description of key information

 

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance, DPHA, using the CO2 evolution (modified Sturm) method according to OECD Guideline 301B, in compliance with GLP. In addition, the procedures were designed to meet EU Method C.4-C, ISO International Standard 9439 (1999) and ISO Standard 10634 (1995). The test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. Since the organic carbon content could not be calculated, a sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC) content. The TOC content of DPHA was determined to be 66%. The test substance was tested in duplicate at 18 mg/l, corresponding to 12 mg TOC/l. Based on the TOC content the ThCO2 of DPHA was calculated to be 2.42 mg CO2/mg. The study consisted of six bottles:(a) 2 inoculum blanks (no test substance) (b) 2 test bottles (DPHA) (c) 1 positive control (sodium acetate) (d) 1 toxicity control (DPHA plus sodium acetate). Weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, approximately 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex), the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29th day). The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of DPHA. In the toxicity control, DPHA was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the test substance was considered as not readily biodegradable (Desmares-Koopmans, 2012).