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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:
July 23, 2015 to December 17, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test Material Name: N-Butyl Propionate
Chemical Name: Butyl ester propanoic acid
Synonyms: Butyl propionate
Lot/Reference/Batch Number: TD13031191
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.93% (Callahan, 2015).
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch at the Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on 27 July 2015. This facility treats an excess of 11 million liters of wastewater per day, of which > 90% is from domestic sources. The activated sludge was collected one day prior to initiation of the test, and was continuously aerated until used. Prior to use, the activated sludge was screened through 500-μm nylon mesh, and briefly homogenized in a Waring blender (Waring Products Inc., Torrington, Connecticut). The mixed liquor suspended solids (MLSS) content of the homogenized sludge was determined gravimetrically to be 3,550 mg/L. Based on this determination, 105 mL of the homogenized activated sludge was added to 12 liters of the sterilized mineral medium to yield a final MLSS concentration of 30.8 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
ThOD
Initial conc.:
21.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
primary indicator of biodegradation
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
supplemental measure of biodegradation
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
supplemental measure of biodegradation
Details on study design:
Justification for Selection of Test System:
This test method is used to determine ready biodegradability by measuring oxygen consumption in a test system consisting of an activated sludge inoculum, test and reference materials, and a defined mineral medium. Further, activated sludge has historically been used to evaluate the persistence of chemicals in the environment. The test system was selected based on the OECD Guidelines (OECD, 1992), and in consideration of the physical/chemical properties of the test material. The test material was expected to be fully soluble in water (water solubility = 1,500 mg/L at 25 °C, USEPA 2012) at the concentration employed in this study. However, experience has shown that the test material will pool on the water surface, and/or form an emulsion/dispersion as it is slowly dissolved in water. The test material has a moderately high vapor pressure (4.4 mm Hg at 25 °C) and is also expected to be moderately volatile from aqueous solution based on its estimated Henry’s Law Constant of 5 x 10^-4 atm*m3/mole at 25 °C (USEPA, 2012). The CES electrolytic respirometer system was employed in this study, as it uses sealed test vessels, continuous stirring, and a relatively small headspace volume which minimize potential for test material loss due to volatilization.

Test Solutions:
The test material was expected to be fully soluble in water (water solubility = 1,500 mg/L at 25 °C, USEPA 2012) at the concentration employed in this study. However, experience had shown that the test material would pool on the water surface, and/or form an emulsion/dispersion as it was slowly dissolved in water. The test material had a moderately high vapor pressure (4.4 mm Hg at 25 °C) and was also expected to be moderately volatile from aqueous solution based on its estimated Henry’s Law Constant of 5 x 10^-4 atm*m3/mole at 25 °C (USEPA, 2012). Therefore, to facilitate precise and efficient transfer of the test material to the biodegradation reaction mixture, it was coated onto a solid carrier (silica gel) and added directly to the biodegradation reaction mixtures on a weight basis (OECD, 1992). Prior to use, the silica gel was fired in a muffle furnace at 550°C to remove any trace organic contaminants. The test material was then coated onto the silica gel (35-60 mesh) and mixed to give a free-flowing homogenous powder. Prior to study initiation, both blank silica and portions of the coated silica were analyzed for total organic carbon (TOC) to verify loading and homogeneity of the test material. The amount of coated silica gel needed to be added to the biodegradation reaction mixtures was determined based on the test material’s theoretical oxygen demand (ThOD). Each test mixture contained 50 - 100 mg/L ThOD as test material. Weighed portions of the coated silica gel were added to the reaction mixtures to yield the required target concentrations of the test material.

Chemicals and Reagents:
De-ionized water used to prepare the mineral medium and reference material stock solutions was purified through a PURELAB Ultra water treatment system (ELGA LabWater, High Wycombe, United Kingdom) producing ultrapure water. All other chemicals used were purchased from commercial sources and had appropriate documentation of identity and purity.

Mineral Medium:
A defined mineral medium was prepared as specified in OECD Guideline 301F, by dissolving appropriate volumes of concentrated mineral stock solutions in ultrapure water (Table 1). The finished mineral medium was filter sterilized with a Corning 0.22 μm filter prior to addition of the inoculum.

Test Procedure:
The biodegradation reaction mixtures were prepared in 0.5-liter glass reaction vessels, each containing a 400-mL portion of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFEcoated magnetic stir bars. All reaction vessels were labeled using a color code/numbering system for vessel identification. Inoculum Blanks, containing the inoculated mineral medium with unamended silica gel without added test or reference material, were prepared in duplicate.
These Inoculum Blanks were used to determine mean values for cumulative O2 consumption, CO2 evolution, and changes in DOC concentration in the absence of added test material. Biodegradation of the reference material, sodium benzoate was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. A concentrated aqueous stock solution was prepared and dispensed into the inoculated mineral medium to give 100.2 mg/L sodium benzoate in the Positive Control mixtures. Biodegradation of the test material in the duplicate Test Suspensions was determined by adding test material-amended silica gel to the inoculated mineral medium (400 mL). A single Toxicity Control reaction mixture was similarly prepared by combining the test material-coated silica gel and aliquot of the sodium benzoate stock solution in the inoculated mineral medium. The rate and extent of sodium benzoate degradation in the Toxicity Control reaction mixture was used to assess whether the test material was inhibitory to the microbial inoculum under the test conditions. Similarly, a single Abiotic Control mixture was prepared by adding mercuric chloride (sterilizing agent, 249 mg/L) to inoculated mineral medium containing the test material-amended silica gel. This Abiotic Control was used to determine the amount of O2 consumption, CO2 evolution, and changes in DOC concentration measured in the Test Suspensions which could be
attributed to abiotic reactions.
After addition of test material, silica gel, sodium benzoate, and sterilizing agent to the appropriate vessels, the pH of the reaction mixtures were measured and adjusted as necessary to 7.4 ± 0.2, then stirred for 30 minutes to homogenize their contents prior to initiation of the test. Samples (30 mL) of the Inoculum Blank, Positive Control, Test Suspensions, Abiotic Control, and Toxicity Control reaction mixtures were collected for initial analyses of dissolved organic carbon (DOC). Specific operating parameters for the respirometer system are described in detail in the study file. The biodegradation reaction mixtures were incubated in the dark at a temperature between 20 and 24 °C. The reaction mixtures were continuously stirred by PTFE-coated magnetic stir bars rotating at a setting of 150 rpm.

Frequency of Sampling:
Concentrations of oxygen in the headspace of each reaction vessel were recorded at six hour intervals over the entire 28 day test period. Upon completion of these measurements on day 28, the DOC concentrations in Inoculum Blank, Positive Control, Test Substance, Abiotic Control, and Toxicity Control reaction mixtures were determined, and TIC concentrations in the caustic traps associated with these reaction mixtures were also determined.

STATISTICS:
Descriptive statistics (mean, standard deviation) were used where applicable.









Reference substance:
benzoic acid, sodium salt
Test performance:
Toxicity Controls:
The Toxicity Control mixture was unable to be correctly analyzed in this study due to the leakage of the test vessel, and resulting malfunction of its manometric sensor of the CES respirometer system. However, analyses of CO2 evolution in the trap of this vessel after 28 days indicated 92.3 % of ThCO2 evolved. Similarly, analyses of DOC removal indicated 100 % of the initial DOC from sodium benzoate and n-butyl propionate was removed. Lastly, the gross O2 consumption in the Test Suspensions was higher than that of the Inoculum Blanks over all measurement intervals of the 28 day test. Collectively, this evidence indicates that the test material was not toxic or inhibitory to the inoculum under the conditions of this test.

Abiotic Controls:
A single Abiotic Control mixture was included in the experimental design to determine the extent to which abiotic processes may result in degradation of the test material. The mixture contained test material in the inoculated mineral medium, which was chemically sterilized by addition of HgCl2. The Abiotic Control mixture exhibited no O2 consumption or CO2 production over the duration of the 28-day test (data not shown). Therefore, the O2 consumption and CO2 production in the Test Suspensions was solely attributed to biodegradation of the test material. The blank-corrected DOC concentration in the Abiotic Control mixture was 11.7 mg/L at test initiation, and was 9.3 mg/L at test termination. Therefore, a small degree of test material DOC removal observed in the Test Suspensions could be attributed to abiotic removal mechanism(s) such as adsorption to biosolids and/or volatilization into the test vessel headspace.
Parameter:
% degradation (O2 consumption)
Value:
69.4
St. dev.:
6.1
Sampling time:
28 d
Details on results:
Biological Oxygen Demand (BOD):
Biological oxygen demand (BOD) is used as the primary indicator of biodegradation in the OECD 301F: Manometric Respirometry test. These measurements of BOD showed the extent of biodegradation of the test substance under the conditions of this test. The time required for average biodegradation to exceed 10% DO2 (i.e., the lag period) was 0.8 days and the 60% DO2 level was exceeded after 8.3 days (See Table 2 in "Any other information on results" section). By the end of the 28-day test, % biodegradation of the test substance reached 69.4 ± 6.1% DO2 (mean ± 1 std. dev., n = 2).
Results with reference substance:
Biodegradation of the reference compound (Sodium Benzoate, 100.2 mg/L) exceeded the pass level within 2.8 days.

Biological Oxygen Demand (BOD):

By the end of the 28-day test, % biodegradation of the test substance reached 69.4 ± 6.1% DO2 (mean ± 1 std. dev., n = 2).

Table 2. Summary of Biodegradation Based on Oxygen Consumption (DO2)

Reaction Mixtures

Time to Achieve (Days)

DO2*(%) at

10% DO2

60% DO2

10-d Window

Day 28

Positive Controls

1.0

2.8

90.1 ± 1.6

97.1 ± 0.0

Test Suspensions

0.8

8.3

63.6 ± 5.1

69.4 ± 6.1

*Mean ± 1std. dev., n = 2

CO2 Evolution:

Two of the OECD tests for ready biodegradability utilize measurements of CO2 evolution to indicate the extent of test substance mineralization. The pass criterion for these tests is 60% of theoretical carbon dioxide evolution. While measurement of CO2 evolution is not a requirement of OECD Guideline No. 301F, these supplemental results for CO2 evolution are consistent with the extent of test substance biodegradation and ready biodegradability conclusion derived from oxygen consumption. Biodegradation of the substance after 28 days reached 66.8 ± 4.6% DCO2 (mean ± 1 std. dev., n = 2).

DOC Analyses:

Because the test material has poor solubility and moderate volatility in the test medium at the concentration evaluated in this test, the extent of biodegradation may not be accurately reflected in analyses of dissolved organic carbon (DOC) removal. However, DOC analyses were performed on the Test Suspensions, Toxicity Control, and Abiotic Control to determine if biodegradation of the test material resulted in formation of any measurable, soluble degradation products. The blank-corrected DOC values in the two Test Suspensions at test initiation indicated that the test material was not fully dissolved under the conditions of this test. The n-butyl propionate substance is composed of 64.6 % carbon, which would give approximately 14 mg/L DOC from the applied 21.6 mg/L test material. The initial blank-corrected DOC concentrations in the duplicate Test Suspensions were 11.1 and 12.0 mg/L, while similarly-determined concentrations after 28 days were 0 (not detected) and 0.1 mg/L. These results for DOC analyses indicate an average 99.4 % removal of the initial DOC over 28 days, and that biodegradation of the test material did not result in generation and accumulation of persistent water-soluble degradation products.

Test Validation:

Several criteria are specified by the OECD for validating the results of its tests for ready biodegradability (OECD, 1992). These criteria are based on parameters such as inoculum viability, precision among replicate reaction mixtures, and maintenance of temperature and pH of the reaction mixtures.

The inoculum used in this test produced > 60% biodegradation of the reference substance, sodium benzoate, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 2.8 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 97.1%, 95.6% and 100.0%, respectively, at the end of the test.

For the Test Suspensions and Positive Controls, the extent of biodegradation recorded for replicate reaction mixtures must not differ by more than 20% DO2 at the end of the 10-day window, plateau of degradation, or the end of the test (OECD, 1992). In this test, the percentage of test substance biodegradation in the replicate Test Suspensions differed by < 9.6% DO2 over all sample intervals of the 28-day test. The maximum difference in percentage of sodium benzoate biodegradation in replicate reaction mixtures was 2.3% DO2 over the course of the study. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.

The recorded temperature in a reference vessel incubated alongside of the biodegradation reaction mixtures gave minimum and maximum temperatures ranging from 22.1 to 22.6 °C over the entire duration of this test. Thus, incubation temperature was maintained within the guideline-required range of 20 - 24 °C and within ± 1 °C variation of the mean incubation temperature.

The pH of the biodegradation reaction mixtures remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Suspensions decreased by no more than 0.22 pH units from their initial values over 28 days, and showed only a 0.19 pH unit (maximum) difference relative to the Inoculum Blanks at the end of the test. This minimal variation in pH indicates that the mineral medium contained adequate buffering capacity for the inoculum and test substances evaluated in this test.

Validity criteria fulfilled:
yes
Remarks:
Biodegradation results of the reference compound confirmed the viability of the inoculum. Other experimental validation parameters fell within the ranges required by the OECD guideline. Therefore, the results of this study are considered fully valid.
Interpretation of results:
readily biodegradable
Conclusions:
The results of this test demonstrate that n-butyl propionate can be classified as “readily biodegradable,” according to the OECD 301F: Manometric Respirometry Test (OECD, 1992). According to the OECD definition of “readily biodegradable,” the substance can be assumed to exhibit rapid and ultimate biodegradation in a variety of environments, including biological sewage treatment plants, surface waters, sediments, and soil.
Executive summary:

The ready biodegradability of n-butyl propionate was evaluated using the OECD Guideline No. 301F: Manometric Respirometry Test. The test employed biodegradation reaction mixtures (400 mL) containing a defined mineral medium, which was inoculated with 30 mg/L activated sludge (dry solids) collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan). Biodegradation of the poorly-soluble and moderately volatile test material was evaluated by coating it onto a silica gel substrate, and addition of this test material-coated silica gel to the reaction mixtures at a concentration of 21.6 mg/L, which was equivalent to approximately 50 mg/L theoretical oxygen demand (ThOD). Oxygen consumption in the biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using a Co-ordinated Environmental Services Ltd. electrolytic respirometer system, which is well-suited for testing of volatile and/or poorly soluble substances. The onset of n-butyl propionate biodegradation (i.e. oxygen consumption ≥ 10% of ThOD) occurred after 0.8 days in the Test Suspensions, and biodegradation exceeded the pass level of 60% ThOD consumption after 8.3 days. At the end of the 28 day test, the extent of biodegradation based on BOD and CO2 evolution reached 69.4 ± 6.1% and 66.8 ± 4.6% (mean ± 1 std. dev., n =

2), respectively. Thus, biodegradation exceeded the pass level within a 10-day window within the 28 day test. No net consumption of oxygen or evolution of carbon dioxide was observed in an Abiotic Control reaction mixture containing 21.6 mg/L of the test material in the inoculated mineral medium, and to which HgCl2 (249 mg/L) was added as a chemical sterilant. Collectively, these results demonstrated that n-butyl propionate meets the OECD definition and criteria for “readily biodegradable” in the Guideline 301F manometric respirometry test.

Biodegradation of the reference compound (Sodium Benzoate, 100.2 mg/L) exceeded the pass level within 2.8 days, and thus confirmed the viability of the inoculum. Other experimental validation parameters, such as reaction mixture pH, incubation temperature, and total oxygen consumption in the Inoculum Blanks, fell within the ranges required by the OECD guideline. Therefore, the results of this study are considered fully valid, and confirming the test material as “readily biodegradable.”

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information