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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Remarks:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Days 3, 6, 8, 10, 14, 17, 22, 27 and 29
Vehicle:
no
Details on test solutions:
The test solutions contained the mineral medium, the test substance suspended at 18 mg/L initial concentration and the activated sludge inoculum obtained from the The concentration of suspended solids was 3.1 g/L in the con
centrated sludge (information obtained from the municipal sewage treatment plant).
Test organisms (species):
activated sludge of a predominantly domestic sewage
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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
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
Nominal and measured concentrations:
Initial test substance concentration 18 mg/L
Details on test conditions:
- 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 spurged 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.
Reference substance (positive control):
yes
Remarks:
Acetic acid, sodium salt
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
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% bio
degradation occurred within 14 days (41%, based on theoretical carbon dioxide). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance (positive control):
The positive control substance was biodegraded by at least 60% (81%) within 14 d.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 28 d NOEC of the test substance in activated sludge microorganisms is 18 mg/L.
Executive summary:

A study was conducted to determine the toxicity to sludge microorganisms 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 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 the test substance 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 (c) 1 positive control (sodium acetate) (d) 1 toxicity control (test substance plus sodium acetate). Weighed amounts were added to the 2 L 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 the test substance. In the toxicity control, the test substance 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 28 d NOEC of the test substance in activated sludge microorganisms is 18 mg/L (Desmares-Koopmans, 2012).

Description of key information

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
18 mg/L

Additional information

A study was conducted to determine the toxicity to sludge microorganisms 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 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 (c) 1 positive control (sodium acetate) (d) 1 toxicity control (test substance 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 the test substance. In the toxicity control, the test substance 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 28 d NOEC of the test substance in activated sludge microorganisms was 18 mg/L (Desmares-Koopmans, 2012).