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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
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)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The activated sludge used for this study was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste. Approximately 4 liters of activated sludge was collected on 26 October 2009 and transported to Springborn Smithers. Upon arrival at Springborn Smithers, the sludge was passed through a 2-mm sieve, combined, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral medium and the contents were centrifuged at least once again and the supernatant was discarded. The moisture content of the activated sludge was determined, using an automated moisture analyzer (Sartorius MA-150), to be 95.7% and the percent solids determined to be 4.30%. A 15 mg solids/mL inoculum solution was prepared (3.0 g dry weight sludge brought to 200 mL with mineral media) and aerated until used. The test substance flasks, the blank flasks, the procedural control flask and the toxicity control flask all received 6.0 mL of the inoculum to produce an activated sludge concentration of 30 mg/L. In addition, a 50-g aliquot of fresh soil and a 50-g aliquot of Weweantic River sediment were collected near Springborn Smithers Laboratories. Upon arrival at Springborn Smithers, the soil and sediment were suspended in 1 L of Weweantic River water. The suspension was filtered through glass wool and refrigerated until use. A 3-mL aliquot of the soil/sediment filtrate was added to each test vessel containing 2991 mL of mineral medium and 6.0 mL of activated sludge
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: theoretical total organic carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Solutions were prepared using reagent grade water (meeting ASTM Type Il requirements) obtained with a Sybron/Barnstead NANOpure II system. The source water for this system is well water that has been shown to be free of all contaminants which could affect study results. The 0.2-μm filtered water typically has greater than 16.7 Mohm-cm resistivity and total organic carbon below the 1 mg/L detection limit (routine monthly analyses). The dissolved organic carbon (DOC) content of the reagent grade water was determined to be 0.16 ppm prior to use. All chemicals were at least reagent grade and were obtained from commercial sources. Each test unit consisted of a 4-L glass bottle with a rubber stopper into which one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were inserted. Prior to test initiation, the test vessels were acid washed and rinsed repeatedly with reagent grade water. The stainless steel needle was extended through the stopper into the test solution serving as a sampling port for solution samples. A rubber cap was used to cover the top of the sample port. The glass tubing provided the inlet and outlet ports for air exchange. CO2-free air was pumped under positive pressure through a hydration flask before entering the test system. The outlet port of each system was connected to two CO2 effluent gas traps, the first consisting of 200 mL of 0.2-N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2-N KOH. Six test vessels were established: two for the test substance, two inoculum blanks, one sodium benzoate procedural control and one toxicity control. On day -1, each 4-L vessel was established by adding 2991 mL mineral medium. A 6.0-mL aliquot of the activated sludge inoculum and 3.0 mL of soil inoculum were added to each vessel for a total volume of 3 L per vessel. All test vessels were attached to a CO2-free compressed air gas tank and aerated under positive pressure. The vessels were mixed and purged with CO2-free air until day 0 to remove any residual inorganic carbon in the test system prior to test initiation. A 10.0 mg C/mL sodium benzoate stock solution was prepared by dissolving 1.7148 g (1.0 g carbon) of sodium benzoate to 100 mL with sterile purified reagent water. This stock solution was used to prepare the procedural and toxicity controls. Since the test substance is not readily soluble in water, it was adsorbed to silica gel prior to dosing to enhance the contact of the test substance with the innoculum. On day 0, approximately 0.0444 to 0.0459 g of the test substance was added to approximately 100 mg of silica gel (Sigma Aldrich Lot No. 10901CH, 230 to 400 mesh, 60Å) for both test suspension vessels and the toxicity control vessel. At test initiation, the toxicity control vessel received 3.0 mL of the 10.0 mg C/mL sodium benzoate stock solution. The total fortification was 20 mg C/L (10 mg C/L test substance and 10 mg C/L reference substance) in the toxicity control vessels. The sodium benzoate procedural control was fortified with 3.0 mL of the sodium benzoate stock solution for a final concentration of 10 mg C/L. The test vessels were identified with at least the project number, test substance, replicate number and treatment type. Each test vessel was placed on a magnetic stir plate located in a dark environmental chamber set to maintain a temperature of 22 ± 2 ºC. All test systems were aerated continuously for 28 days under positive pressure using CO2-free air in order to provide oxygen for the microbes and to capture evolved carbon dioxide. The temperature of the environmental chamber was recorded throughout the exposure period using a digital VWR minimum-maximum thermometer.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
73.05
St. dev.:
4.99
Sampling time:
28 d
Details on results:
The 28 day cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, for Eastman 168 Plasticizer (terephthalate), procedural control, and toxicity control was calculated to be 73.05, 78.91 and 77.91%, respectively. Based upon a graphical display of the CO2 evolution data for the test substance, the 10 day window for achieving 60% degradation within 10 days after achieving 10% degradation was met. The cumulative net CO2 evolved from the sodium benzoate procedural control was 65.91% of theoretical by day 10 confirming the presence of an active microbial population and system integrity.
Results with reference substance:
The cumulative net CO2 evolved from the sodium benzoate procedural control was 65.91% of theoretical by day 10. This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the CO2 analysis results from this study, Eastman 168 Plasticizer is "readily biodegradable" according to the OECD 301B guideline. The rapid degradation of the reference substance confirmed the presence of an acceptable microbial community and confirmed system integrity.
Executive summary:

The determination of the biodegradability of Eastman 168 Plasticizer based on OECD Method 301B (CO2 Evolution Test) was conducted. The cumulative net percent CO2production (blank control values subtracted), or percent ultimate biodegradation, for Eastman168 Plasticizer, procedural control, and toxicity control was calculated to be 73.05, 78.91 and 77.91%, respectively. The rapid degradation of the reference substance confirmed the presence of an acceptable microbial community and confirmed system integrity. Based on the CO2 analysis results from this study, Eastman168 Plasticizer is "readily biodegradable" according to the OECD 301B guideline.

Description of key information

Two OECD 301B Ready Biodegradation Studies

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

In 1985 an OECD 301B ready biodegradation study was conducted with Eastman 168 Plasticizer ( Bis (2-ethylhexyl) terephthalate). The results of that study indicated significant degradation occurred (40.2%), but not enough to classifiy the substance as readily biodegradable. After review of the degradation potential of the substance, it was believed that it was likely that there may be an issue with the low solubility of the substance reducing the bioavailability to the test micro-organisms in the study. A second OECD 301B study was conducted in 2009 where the substance was introduced into the test system through application to fine silica gel to increase the surface area and bioavailability of the substance to the microorganisms. This study resulted in 73.05% degradation over 28 days and meeting the 10 day window criteria. Therefore, the test substance is considered to be "readily biodegradable".