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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 301B, EPA OPPTS 835.3110, EU Method C.4-c and in accordance with the Principles of Good Laboratory Practice (GLP)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Remarks:
Minor exceptions eg. non availability of expiration date for the test substance and characterization of the reference substance (sodium benzoate) not conducted under GLP, however, these did not impact the overall integrity or the outcome of the study
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: dark green powder
- Physical state: solid
- Analytical purity: ~ 97% active ingredient
- Lot/batch No.: Lot #020801; TD No. 02-045
- Stability under test conditions: expected to be stable for the duration of testing
- Storage condition of test material: room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge - The microbial inoculum was an activated sludge mixed liquor collected. The activated sludge was collected at the Columbia Wastewater Treatment Plant in Columbia, Missouri, which predominantly treats domestic sewage. Approximately 0.5 liter of activated sludge collected from aeration basin #1 was used for this study.

The activated sludge was homogenized in a blender at a medium speed for two minutes. The homogenized sludge was allowed to settle for 30 to 60 minutes, filtered through glass wool, and then aerated with COz-free air until use. Thirty milliliters of the prepared activated sludge was used as the inoculum for each reaction flask.

The suspended solids concentration in the prepared activated sludge was determined by filtering three 10-mL aliquots of sludge through pre-weighed glass-fiber filter pads, followed by drying on a Mettler HR73P halogen moisture analyzer. The increase in weight of the filter pads was used to determine the suspended solids level. The suspended solids concentration was 100 mg/L in the prepared mixed liquor suspended solids. Therefore, the total concentration of suspended solids in each reaction flask (30 mL of inoculum to 3000 mL of test medium) was 1 mgIL.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
>= 1.93 - <= 4.53 other: C/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Each test system consisted of a 5-L glass flask (reaction flask) containing a 3-L test solution volume comprised of mineral medium, microbial inoculum, reagent water, and the appropriate test or reference substance additions. To remove CO2, the incoming air was passed through a column containing Ascarite followed by a pre-trap containing 500 mL of approximately 5 N KOH. The air was then passed through 500 mL of reagent water to re-humidify the air, as well as to prevent contamination of the flasks from the KOH pre-trap. The CO2-free and humidified air was then passed through the reaction flasks.
Air was introduced into each flask by positive pressure, and the flow rates (50-100 mllminute) were measured and adjusted using flow meters. The outlet from each flask was connected to three CO2 absorber gas-washing traps in series, each filled with 100 mL of 0.2 N KOH solution (Figure I). These traps captured theCO2 evolved from the reaction flasks. A magnetic stir bar was placed in each flask. The flasks were placed on insulated magnetic stir plates and stirred throughout the duration of the study. The test systems were kept in the dark (except for sampling and maintenance) in a temperature controlled environmental chamber maintained at 22 + 2°C. Temperature of the chamber was continuously measured using a bi-metallic (aluminum-constantan) thermocouple probe and Multiscan 1200 temperature monitoring system.
The test substance, which was described as totally insoluble in water, was weighed on glass coverslips, which were added directly to the appropriate reaction flasks.
A 1.0 mg/mL stock solution of the reference substance was prepared by weighing 1007 mg of sodium benzoate, correcting for purity (99.3%), and diluting in 1000 mL of reagent water. The solution was designated as CDG 2952 and was refrigerated when not in use.
Duplicate control systems were prepared by adding 570 mL of reagent water to 5-L carboys with the final volume of 3000 mL. Duplicate test substance systems were prepared by adding 570 mL of reagent water and a glass coverslip containing approximately 76.0 mg of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) to 5-L carboys. The measured weights of test substance were 76.2 and 76.1 mg for replicates 1 and 2, respectively. The nominal concentration of carbon from the test substance in the final volume of 3000 mL of solution was 20.1 and 20.0 mg C/L, respectively.
The reference substance system was prepared by adding 467 mL of reagent water and 103 mL of the 1.0 mg/mL reference substance stock solution (CDG 2952) to a 5-L carboy. The nominal concentration of carbon from the reference substance in the final volume of 3000 mL of solution was 20.0 mg C/L.
The toxicity control system was prepared by adding 467 mL of reagent water, 103 mL of the 1.0 mg/mL reference substance stock solution (CDG 2952), and a glass coverslip containing 76.2 mg of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) to a 5-L carboy. The total nominal concentration of carbon from the reference substance and 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) in the final volume of 3000 mL of solution was 40.1 mg C/L (i.e., 20.0 mg C/L from the reference substance and 20.1 mg C/L from the test substance).
The abiotic sterile control system was prepared by adding 570 mL of reagent water, a glass coverslip containing 76.1 mg of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ), and 150.5 mg of mercuric chloride (HgC12) to a 5-L reaction flask. The nominal concentration of carbon from the test substance in the final volume of 3000 mL of solution was 20.0 mg C/L. After all additions, each of the reaction flasks was connected to a series of three gas-washing bottles containing 100 mL of 0.2 N KOH. Aeration and stirring of the flasks were continued. Flow meters connected to the test systems were adjusted to facilitate air flow at 50-1 00 mL/minute. The bubbling of air and stirring in each flask, as well as the bubblingin each gas-washing bottle, confirmed the constant aeration.
Approximately 45 minutes after dosing, the pH of each test solution was measured, and approximately 100 mL of each test solution was removed and filtered through 0.45-µm nylon syringe filters. Each filtrate was deposited in a 2-oz. amber bottle and three 14-mL TOC autosampler vials. The bottles and vials were filled leaving no headspace, capped, and stored refrigerated until analysis. Filtrates in the 2-oz. bottles were analyzed for dissolved organic carbon (DOC) concentration, and filtrates in the 14-mL vials were analyzed for inorganic carbon (IC) concentration.
The CO2 produced in the test systems was trapped in the 0.2 N KOH solutions, which were then analyzed for inorganic carbon (IC) content. Samples of the KOH solutions were collected for CO2 analysis on days 3, 5, 8, 11, 17, 21, 25, 28, and 29. For each sample day except day 29, triplicate aliquots of the KOH solution from the gas-washing bottle nearest each flask were placed into appropriately-labeled glass autosarnpler vials. The vials were filled leaving no headspace, capped using Teflon septa, and stored at room temperature until analysis. The first replicate of each set of triplicate samples was analyzed for IC content; and the other two were used as reserve samples. The remaining KOH solution in this gaswashing bottle was discarded and replaced with 100 mL of a fresh 0.2 N KOH solution. The refilled gas-washing bottle was then rotated to the position farthest from the flask, and the other two gas-washing bottles were moved forward (nearer to the flask) one position.
After sampling the KOH solutions on day 28, approximately 60 mL of each test solution was removed and filtered through 0.45-µm nylon syringe filters. Each filtrate was deposited in a 2-oz. amber bottle, which was filled leaving no headspace, capped, and stored refrigerated until analysis for dissolved organic carbon (DOC) concentration. The pH of the test solutions was measured, and aliquots of the test solutions were removed for bacterial plate count analyses. After sampling the test solutions, 1 mL of concentrated HCl was added to each test solution to drive carbonates and the remaining C02 from solution. The flasks were then re-sealed and allowed to aerate overnight. On day 29, triplicate aliquots of each gas-washing bottle were taken for IC analysis, and the gas-washing bottles were not refilled with 0.2 N KOH.
Bacterial plate counts were performed for the prepared activated sludge and samples of the test solutions collected on day 28. Aliquots from the duplicate systems (i.e., control and test substance) were pooled prior to analysis. Dilutions of each sample were prepared in sterile, pH 7.2, phosphate-buffered water. Duplicate 1-mL aliquots of each dilution were directly analyzed by plate counting methods patterned after methods described in Standard Methods for the Examination of Water and Wastewater. The bacterial growth medium was Plate Count Agar. The plates were incubated at 26 + 2°C for three to four days before counting the number of colonies on plates with fewer than 300 colonies. The number of colonies at the dilution coming closest to 300 colonies was used to calculate colony forming units (CFU)/mL for each sample.




Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
(Lot No. A0142142, purity 99.3%) from Acros Organics

Results and discussion

Preliminary study:
not applicable
Test performance:
not applicable
% Degradation
Sampling time:
29 d
Remarks on result:
other: The percent theoretical CO2 produced by 1,4,5,8-Tetra (4'-n-butylphenylarnino) Anthraquinone (TBPAAQ) was 2.4% and 2.1% ThCO2 by day 29 of the study, hence, TBPAAQ cannot be classified as readily biodegradable.
Details on results:
The pH of the control solutions was 7.58 and 7.60 at study initiation and 7.54 and 7.51 at termination for replicates 1 and 2, respectively. The pH of the controlsolutions was approximately the pH of the mineral medium, 7.60, indicating that the mineral medium adequately buffered the pH after addition of the inoculum. The pH of the test substance solutions decreased from 7.60 and 7.61 at study initiation to 7.50 and 7.48 at study termination for replicates 1 and 2, respectively. The pH of the abiotic sterile control system also decreased and was 7.39 and 7.31 at initiation and termination, respectively. The pH of the reference substance system increased from 7.62 at study initiation to 7.73 at study termination. The pH of the toxicity control solution increased from 7.60 at study initiation to
7.69 at termination. The decrease in pH for the reference substance and toxicity control solutions is typical for solutions containing biodegradable substances. All pH values were suitable for biological systems.
The temperature of the environmental chamber ranged from 21.5"C to 22.1°C during the 29-day test. The mean and standard deviation of the temperature measurements was 21.8 ± 0.1 °C.
At study initiation, the DOC concentration of the control solutions was 2.22 and 3.23 mg C/L for replicates 1 and 2, respectively. At study termination, the DOC concentration of the control solutions was 1.87 and 1.90 mg C/L for replicates 1 and 2, respectively.
The DOC concentrations of the test substance solutions at initiation were 4.53 and 1.93 mg C/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the controls (2.73mg C/L), DOC concentrations were 1.81 and < 0 mg C/L, corresponding to 9% and 0% of the nominal 20.1 and 20.0 mg C/L testing concentrations. The low recoveries confirm that the water solubility of the test substance was lower than the nominal test concentration (20 mg C/L or 24.6 mg/L). At termination, the DOC concentrations of the test substance solutions were 1.82 and 1.88 mg C/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the controls at termination (1.89 mg C/L), DOC concentrations in the test substance solutions were < 0 mg C/L.
The DOC concentration of the abiotic sterile control solution at initiation was 2.75 mg C/L. Correcting for the mean DOC concentration measured in the controls (2.73 mg C/L), DOC concentration was 0.02 mg C/L, corresponding to 0% of the nominal 20.0 mg C/L testing concentration. The low recovery confirms that the water solubility of the test substance was lower than the nominal test concentration (20 mg C/L or 24.6 mg/L). At termination, the DOC concentration of the abiotic sterile control solution was 2.15 mg C/L. Correcting for the mean DOC concentration measured in the controls at termination (1.89 mg C/L), DOC concentration in the abiotic sterile control solution was 0.27 mg C/L.
The DOC concentration of the reference substance solution at initiation was 25.4 mg C/L. Correcting for the mean DOC concentration measured in the controls (2.73 mg C/L), the DOC concentration was 22.7 mg C/L, corresponding to 113% of the nominal 20.0 mg C/L testing concentration. The acceptable recovery result (70-120%) indicated that the reference substance system was prepared correctly. At termination, the DOC concentration of the reference substance solution was 2.06 mg C/L. Correcting for the mean DOC concentration measured in the controls at termination (1.89 mg C/L), the DOC concentration in the reference substance solution was 0.18 mg C/L.
The DOC concentration of the toxicity control solution at initiation was 23.9 mg C/L. Correcting for the mean DOC concentration measured in the controls
(2.73 mg C/L), the DOC concentration was 21.2 mg C/L, corresponding to 53% of the nominal 40.1 mg C/L testing concentration. The recovery result indicated that the reference substance was correctly added to the toxicity control system and confirmed that the test substance was insoluble in water. At termination, the DOC concentration of the toxicity control solution was 2.10 mg C/L. Correcting for the mean DOC concentration measured in the controls at termination (1.89 mg C/L), the DOC concentration in the toxicity control solution was 0.22 mg C/L. The DOC concentration of the reagent water used in this test was 1.02 mg C/L.
At study initiation, the IC concentrations of the control solutions were 0.714 and 0.934 mg C/L for replicates 1 and 2, respectively. The IC concentrations of the test substance solutions at initiation were 0.844 and 1.05 mg C/L for replicates 1 and 2, respectively, corresponding to 4% and 5% of the nominal initial DOC concentrations, 20.1 and 20.0 mg C/L, for replicates 1 and 2, respectively. The abiotic sterile control solution contained 0.844 mg IC/L at initiation, corresponding to 4% of the nominal initial DOC concentration, 20.0 mg C/L. The IC concentration of the reference substance solution at initiation was 0.704 mgC/L or 4% of the nominal initial DOC concentration, 20.0 mg C/L. The toxicity control solution contained 0.634 mg IC/L at initiation, corresponding to 2% of
the nominal initial DOC concentration, 40.1 mg C/L. The IC concentrations were within the protocol-defined limit (<5% of the nominal concentration) except for replicate 2 of the test substance solutions for which the IC concentration was 5% of the nominal DOC concentration.
The bacterial plate counts showed that the prepared activated sludge filtrate contained 2.3 x 10(6) colony forming units (CFU)/mL. Bacterial plate counts at termination showed that the control solutions contained a mean of 3.4 x 10(5) CFU/mL. The test substance solutions contained a mean of 3.9 x 10(5) CFU/mL. The reference substance solution contained 3.9 x 10(5) CFU/mL at termination. Bacterial plate counts from the toxicity control treatment were 9 x 10(2) CFU/mL and may indicate that the test substance was toxic to the microbes. However, the bacterial plate counts of the test substance treatment (3.9 x 10(5) CFU/mL) show that the test substance was not toxic. These microbial evaluation data suggest that the microbial populations in the test and reference substance systems were active and viable.
CO2 evolved from the control systems was 31.3 and 25.2 mg CO2 for replicates 1 and 2, respectively, by day 29 of the study. These values were corrected for the background CO2 present in the fresh KOH solutions. The goal of the control systems was to provide the background CO2 values from the endogenous CO2 evolution from the microbial inoculum. The total mg CO2 evolved from the controls (31.3 and 25.2 mg CO2/flask) was within the limits indicated in the protocol (<40 mg CO2/L or < 20 mg CO2/flask).
TBPAAQ exhibited final % ThC02 values (after correction for mean background CO2 from the controls) of 2.4% and 2.1% for replicates 1 and 2, respectively, through day 29 of the study. The % ThCO2 values for the test substance solutions did not reach 10% ThCO2. Therefore, 1,4,5 ,%Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) cannot be classified as readily biodegradable according to the criteria outlined in the testing guidelines (60% ThCO2 within a 10-day window after reaching 10% ThCO2). Biodegradation of the test substance based on DOC measurements of the reaction solutions at initiation (day 0) and termination (day 28) could not be determined because the water solubility of the test substance was less than the testing concentration.
The abiotic sterile control treatment exhibited a final % ThC02 value of 12.5% through day 29 of the study. The % ThCO2 values in the abiotic sterile treatment were corrected for the background CO2 present in the fiesh KOH solutions. Data from the abiotic sterile control and the test substance biodegradation treatments show that CO2 production in the test substance systems may not be attributed to biodegradation. Degradation of the test substance based on DOC measurements of the reaction solutions at initiation (day 0) and termination (day 28) could not be determined because the water solubility of the test substance was less than the testing concentration.
The toxicity control, containing readily biodegradable sodium benzoate and 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ), exhibited a % ThCO2 value of 65.6% by day 29 of the study. DOC measurement of the toxicity control solution at initiation (day 0) and termination (day 28) showed that 99% of the DOC was removed by biodegradation. Since biodegradation was greater than 25% based on CO2 production and greater than 35% by DOC removal, the toxicity control treatment proved that 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was not inhibitory to the microbial inoculum at the testing concentration according to the testing guidelines. The toxicity control further indicates that the low level of biodegradation in the test substance
systems was not caused by microbial inhibition.

BOD5 / COD results

Results with reference substance:
Sodium benzoate exhibited a % ThCO2 value of 107% by day 29 of the study. The results from day 3 (61.0% ThCO2 evolved) indicated greater than 60% ThCO2 evolved in the first three days of the test. These results indicate that the inoculum was viable according to the criteria outlined in the testing guidelines. Biodegradation of the reference substance based on DOC measurements of the reaction solution at initiation (day 0) and termination (day 28) was 99%' confirming the measured biodegradation from CO2 evolution.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
Under the conditions of the study, the percent theoretical CO2 produced by 1,4,5,8-Tetra (4'-n-butylphenylarnino) Anthraquinone (TBPAAQ) was 2.4% and 2.1% ThCO2 by day 29 of the study. Therefore, 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) cannot be classified as readily biodegradable.
Executive summary:

The objective of this study was to measure the extent of biodegradation of 1, 4, 5, 8-Tetra (4'- nbutylphenylamino) Anthraquinone (TBPAAQ) when exposed to a microbial inoculum in an aerobic, mineral salts medium at an initial test concentration of 20 mg carbon/liter (mg C/L). The inoculum was composed of activated sludge suspension. Duplicate control systems, containing the microbial inoculum with no test or reference substance, were used to determine the endogenous microbial CO2 evolution.

Duplicate inoculated test substance systems, which were dosed with the test substance at nominal concentrations of 20.1 and 20.0 mg C/L, were used to monitor biodegradation of the test substance. A reference substance system containing readily biodegradable sodium benzoate at a nominal concentration of 20.0 mg C/L was tested to verify the viability of the microbial inoculum. A toxicity control containing readily biodegradable sodium benzoate at a nominal concentration of 20.0 mg C/L and 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) at a nominal concentration of 20.1 mg C/L was tested to evaluate toxicity of the active ingredient. An abiotic sterile control system that included the microbial inoculum, test substance (20.0 mg C/L), and a sterilant (HgC12 at 50 mg/L) was used to monitor abiotic degradation of the test substance. All systems were sampled for CO2 trapped in 0.2 N KOH on days 3, 5, 8, 11, 17, 21, 25, 28, and 29. The 0.2 N KOH trapping solutions were analyzed for CO2 by inorganic carbon analysis on a total organic carbon analyzer. The average CO2 evolved from the control systems was subtracted from the CO2 evolved in the test and reference substance systems.

The percent theoretical CO2 (% ThCO2) evolved for the test substance systems was 2.4% and 2.1% for replicates 1 and 2, respectively, by day 29 of the study. The results from the abiotic sterile control system indicated that CO2 production in the test substance systems may not be attributed to biodegradation since abiotic degradation reached 12.5% ThCO2 by day 29. Since biodegradation did not reach 60% ThCO2, the test substance, 1,4,5,8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ), cannot be classified as readily biodegradable (>60% ThCO2 ten days after reaching 10% ThCO2) according to the criteria outlined in the testing guideline. The percent theoretical CO2 produced by the reference substance, sodium benzoate, was 61 .0% by day 3 and 107% by day 29, proving that the inoculum was viable. The percent theoretical CO2 produced from the toxicity control system was 65.6% by day 29, indicating that 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was not inhibitory to the microbial inoculum at the concentration tested.