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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:
April-July 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)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: F-3014
Lot/Batch Number: 290110574
CAS Number: Not Given
Physical Description: Solid
Purity: Not Given
Expiration Date: Not Given
Storage Conditions: Ambient
Carbon Content: 31.14% (as determined by elemental analysis)
Oxygen conditions:
aerobic
Remarks:
aerated with CO2-free air.
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Activated sludge was collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on April 19, 2012. The Cambridge facility treats predominantly residential wastes. The sludge was sieved using a 2-mm screen, adjusted to approximately 1,000 mg total suspended solids/L with mineral media and then aerated at test temperature until use. A total suspended solids measurement and standard plate count were performed on the inoculum on the day of use in the test. Plates were incubated at 20±1ºC for approximately 48 hours.
Duration of test (contact time):
28 d
Initial conc.:
ca. 10 other: mg C/L
Based on:
IC (inorganic carbon)
Remarks:
nominal concentration of 10 mg C/L were added
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test Substance
substance used in this study:
Name: F-3014
Lot/Batch Number: 290110574
CAS Number: Not Given
Physical Description: Solid
Purity: Not Given
Expiration Date: Not Given
Storage Conditions: Ambient
Carbon Content: 31.14% (as determined by elemental analysis)
The test substance was administered to the treatment group test chambers by direct weight addition. Direct weight addition of a test substance that is relatively insoluble in water is the most appropriate route of administration. The amount of test substance used to dose the treatment group test chambers was calculated based on the measured carbon content of the test substance, which is comprised of four components. The carbon content of the test substance was determined by a non-GLP elemental analysis performed by Quantitative Technologies, Inc.

Reference Substance
Name: Sodium benzoate
Manufacturer: Fluka
Lot Number: 1373379
Physical Description: Solid
Chemical Abstract Number: 532-32-1
Retest Date: December 2013
Purity: ≥99.5%
Storage Conditions: Ambient
A stock solution of the reference substance was prepared in NANO pure water at a nominal carbon concentration of 400 mg C/L one day prior to use in the test and stored in refrigerated conditions. The stock solution was analyzed for total organic carbon (TOC). The reference substance was administered to the reference group test chambers by volumetric addition. Dosing amounts were based on the measured carbon content of the reference substance solution as determined using a Shimadzu Model TOC-VCSH carbon analyzer.

Test Medium
The test medium was a modified biochemical oxygen demand (BOD) test dilution water and
was prepared using high quality water. All chemicals and reagents used in the preparation of the test medium were reagent grade or better.

Test Apparatus and Conditions
The test chambers were amber 4-liter bottles. The air entering the chambers was passed through Drierite™ to remove ambient moisture and then through Ascarite® to produce CO2-free air. The air exiting the test chambers was passed through a series of three gas washing bottles, each containing approximately 100 mL of 0.5 M KOH to trap the CO2 that had evolved within the chamber. An additional set of gas washing bottles that was not connected to a chamber was maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 M KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the control traps to determine the amount of CO2 produced by the inoculum in the blank control. The test was conducted at 20±1ºC. Test chambers were identified by project number, test substance ID, test concentration and vessel number. Magnetic stir bars and stir plates were used to mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 15 minutes to prevent the heating of the stirrer motors.

Test Inoculum
Activated sludge was collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on April 19, 2012. The Cambridge facility treats predominantly residential wastes. The sludge was sieved using a 2-mm screen, adjusted to approximately 1,000 mg total suspended solids/L with mineral media and then aerated at test temperature until use. A total suspended solids measurement and standard plate count were performed on the inoculum on the day of use in the test. Plates were incubated at 20±1ºC for approximately 48 hours.

Preparation of Test Chambers
The following were added to each chamber:
1) 2470 mL of NANO pure water
2) 3 mL calcium chloride solution (2.75%)
3 mL of ferric chloride solution (0.025%)
3 mL of magnesium sulfate solution (2.25%)
30 mL of phosphate buffer (pH 7.4)
3) A volume of activated sludge inoculum to achieve a final TSS concentration of
≤30 mg/L
All chambers were aerated with CO2-free air for approximately 24 hours at a rate of 50 to 100 mL per minute to purge the systems of CO2. After the aeration period, the flow of CO2-free air was stopped, and three CO2 traps, each containing approximately 100 mL of 0.5 M KOH, were connected to the exit air lines of each chamber. Sufficient volumes of reference substance stock solution to achieve a nominal concentration of 10 mg C/L were added to the appropriate chambers. Sufficient amounts of test substance to achieve a nominal concentration of 10 mg C/L were added to the appropriate chambers. The final volume within all chambers was brought up to 3000 mL by the
addition of NANO pure water.

Biodegradation Test Initiation
The biodegradation test was started by bubbling CO2-free air through each of the test chambers at a rate of approximately 60 mL per minute. The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as K2CO3 in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study, using a Shimadzu Model TOC-VCSH carbon analyzer.
Reference substance:
benzoic acid, sodium salt
Remarks:
Name: Sodium benzoate Manufacturer: Fluka Lot Number: 1373379 Physical Description: Solid CAS Number: 532-32-1 Retest Date: December 2013 Purity: ≥99.5% Storage Conditions: Ambient
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
not measured/tested
Remarks:
St. dev.
Details on results:
Carbon Analysis
The measured total organic carbon (TOC) concentration of the reference substance stock solution was 404.9 mg C/L. The volume of stock solution used to dose the reference chambers was adjusted based on the measured TOC value so that approximately 10 mg C/L was delivered.

Observations and Measurements
The temperature range recorded during the test was 19.3-20.8ºC and was within the protocol specified range throughout the test. The average TSS of the activated sludge on the day of collection was 12,687 mg/L. The results of the standard plate count and TSS measurement performed on the inoculum were 1.1 x106 CFU/mL and 900 mg/L, respectively.
The control chambers evolved an average of 65.0 mg CO2 over the test period. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the blank control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2 evolution tests.
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 101.8% of theoretical CO2 was evolved. An average percent biodegradation of greater than 60% was achieved by Day 6, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 14. The final mean percent biodegradation for F-3014 was –1.2%. F-3014 may not be considered readily biodegradable, since the pass level of 60% ThCO2 was not achieved.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% ThCO2 within the 28-day test period (1,2). In addition, the pass level must be reached within 10 days of achieving 10% ThCO2. The final mean percent biodegradation for F-3014 was –1.2%. F-3014 may not be considered readily biodegradable since the pass level of 60% ThCO2 was not achieved.
Executive summary:

The ready biodegradability of F-3014 was determined by the Carbon Dioxide Evolution Test Method (OECD Guideline 301B). Tests of ready biodegradability are stringent tests that provide limited opportunity for acclimation and biodegradation to occur. In the CO2 test, inoculated mineral

medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air. The CO2 produced from the mineralization of organic carbon within the test chambers was displaced by the flow of CO2-free air and trapped as K2CO3 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2 (ThCO2) that could have been produced if complete biodegradation of the test substance occurred. The test contained a blank control group, a reference group and a treatment group. Each group contained three replicate test chambers. The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group test chambers were used to evaluate F-3014 at a nominal concentration of 10 mg C/L.

The results indicated that the activated sludge inoculum was active, degrading the reference substance an average of 101.8%. The average cumulative percent biodegradation for F-3014 was -1.2%.

Description of key information

Test Substance: F-3014
Average Cumulative Percent Biodegradation: -1.2
Average Final pH: 7.3

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of F-3014 was determined by the Carbon Dioxide Evolution Test Method (OECD Guideline 301B). The test contained a blank control group, a reference group and a treatment group.

The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group test chambers were used to evaluate F-3014 at a nominal concentration of 10 mg C/L.

The results indicated that the activated sludge inoculum was active, degrading the reference substance an average of 101.8%. The average cumulative percent biodegradation for F-3014 was -1.2%.

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