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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:
2004-11-17 to 2005-01-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3120 (Sealed Vessel Carbon Dioxide Production Test)
Qualifier:
according to guideline
Guideline:
other: ISO/DIS-14593 Headspace and ASTM E1720-95 Sealed Vessel
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge: New Bedford Wastewater Treatment Facility, New Bedford, Massachusetts and Wareham Wastewater Treatment Plant, Wareham, Massachusetts, USA
Soil: Wooded area (2004-11-17) near testing facility
- Laboratory culture: No
- Storage conditions: Not stored
- Preparation of inoculum for exposure:
Activated sludge: Collected on 2004-11-16. Upon arrival the sludges were passed through a 2 mm stainless steel metal screen, combined and then centrifuged in four 1L bottles for 10 minutes at 1500 rpm. The supernatant from each bottle was decanted and the sludge kept on bottles. Tap water was used to wash the sludge.
Soil: The soil was passed through a 2 mm stainless steel metal screen and suspended in 1L of tap water. The suspension was filtered through glass wool and refrigerated until use.
- Pretreatment: No
- Concentration of sludge: sludge: 10 mg solids/L, soil: 1 mL soil filtrate/L activated sludge innoculum.
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Phosphate buffer 10 mL stock/L medium, and 1 mL stock/L medium of the following; calcium chloride, magnesium sulphate and ferric chloride
- Additional substrate: None
- Solubilising agent (type and concentration if used): Not used
- Test temperature: 17.7 - 22.5°C
- pH: Not examined
- Aeration of dilution water: No
- Suspended solids concentration: 10 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 20 mL crimp cap vials containing 13.5 mL aqueous test medium
- Number of culture flasks/concentration: 27
- Method used to create aerobic conditions: headspace gas only
- Measuring equipment: Dohrmann DC-80 TOC Analyzer and Thermoglas Model 1200 Carbon Analyzer
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics if used: Not required


SAMPLING
- Sampling frequency: Days, 2, 4, 7, 10, 14, 21 and 28
- Sampling method: Test vessel acidified by injection of 100µL 4.9M phosphoric acid then shaken at 250 rpm for at least 60 mins before analysis.
Determination of DOC: 2 drops of phosphoric acid used to preserve test solutions after sampling and filtering through a 0.45 µm Gelman filter. 1 mL solution injected into analyzer.
Determination of total inorganic carbon: 200 µL headspace gas from acidified test vessels injected into analyzer and results compared with calibration curve from at least 5 concentrations of sodium bicarbonate
- Sterility check if applicable: No
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 13.5 mL of innoculated test medium only
- Abiotic sterile control: No
- Toxicity control: Toxicity reference substance sodim benzoate at 10 mgC/L


STATISTICAL METHODS: None
Amount of carbon dioxide evolved from test system adjusted by subtracting the CO2 production value from the blank control.
% biodegradation = mg CO2 produced/(mg TOC added x 3.67) x 100
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not performed
Test performance:
Test validity criteria fulfilled.
Parameter:
% degradation (CO2 evolution)
Value:
18
St. dev.:
1.07
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
66.5
St. dev.:
0.75
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
73.1
St. dev.:
5.66
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
70.17
St. dev.:
2.76
Sampling time:
28 d
Details on results:
Day 0 dissolved organic carbon (DOC) results of the test solution produced values of 1.0, 9.3 and 11.5 mg DOC/L for the innoculum blank, test material solution and the reference solution, respectively. These results show that the test material solution and the reference solution were near the nominal concentration of 10 mg DOC/L.
The reference substance, sodium benzoate, evolved 93.5% CO2 over the 28-day period and exceeded 60% CO2 evolution within the first two days, thus validatinng the test and indicating the satisfactory viability of the inoculum. In addition, the rapid and extensive biodegradation of sodium benzoate confirmed the presence of an active and viable microbial population and confirmed system integrity.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Approximately 10% CO2 was evolved by Day 1 and approximately 60% CO2 was evolved by day 11, thereby indicating the test material is "readily biodegradable".
The reference substance, sodium benzoate, was considered readily biodegradable under the OECD definition (> 60% CO2 production in 28 days and within a 10-day window of reaching 10% CO2 production). Thus the microbial inoculum for the study was acceptable.
Executive summary:

The study was performed to determine the potential for biodegradation of the test material in water by the CO2 evolution method based on ASTM 1720 -95 Sealed Vessel, the ISO/DIS-14593 Headspace and the OPPTS 835.3120 CO2 evolution biodegradation test guidelines. A mixed activated sewage sludge and soil inoculm was used. The test material and the reference substance were added to the inoculum at a concentration of 10 mg C/L. A blank control containing inoculum only was run alongside the test material vessels. Test vessels are incubated aerobically in the dark for a period of 28 days at ca. 22°C.

Approximately 10% CO2 was evolved by Day 1 and approximately 60% CO2 was evolved by day 11, thereby indicating the test material is "readily biodegradable".

The reference substance, sodium benzoate, was considered readily biodegradable under the OECD definition (> 60% CO2 production in 28 days and within a 10-day window of reaching 10% CO2 production). Thus the microbial inoculum for the study was acceptable.

Description of key information

The substance was readily biodegradable in a test performed according to ASTM 1720 -95 Sealed Vessel, the ISO/DIS-14593 Headspace and the OPPTS 835.3120 CO2 evolution biodegradation test guidelines.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The study was performed to determine the potential for biodegradation of the test material in water by the CO2 evolution method based on ASTM 1720 -95 Sealed Vessel, the ISO/DIS-14593 Headspace and the OPPTS 835.3120 CO2 evolution biodegradation test guidelines. A mixed activated sewage sludge and soil inoculm was used. The test material and the reference substance were added to the inoculum at a concentration of 10 mg C/L. A blank control containing inoculum only was run alongside the test material vessels. Test vessels are incubated aerobically in the dark for a period of 28 days at ca. 22°C.

Approximately 10% CO2 was evolved by Day 1 and approximately 60% CO2 was evolved by day 11, thereby indicating the test material is "readily biodegradable".

The reference substance, sodium benzoate, was considered readily biodegradable under the OECD definition (> 60% CO2 production in 28 days and within a 10-day window of reaching 10% CO2 production). Thus the microbial inoculum for the study was acceptable.