2-(3-oxazolidinyl)ethyl methacrylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 22, 2016 to December 7, 2016

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: Oxazolidinyl Ethyl Methacrylate
Chemical Name: 2-Methyl-2-propenoic acid 2-(3-oxazolidinyl)ethyl ester
Synonyms: OXEMA, 2-(3-Oxazolidinyl)ethyl methacrylate
Lot/Reference/Batch Number: R03NA03
Purity/Characterization (Method of Analysis and Reference): The test material was determined to have a purity of 94.6% by gas chromatography with identification by nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry (Megregian, 2016).

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum:
The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on May 12, 2016. 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 1,527 mg/L. Based on this determination, the homogenized activated sludge was added to the sterilized mineral medium to yield a final MLSS concentration of 29.6 mg/L (dry wt.).

Duration of test (contact time):

28 d

Initial conc.:

48.3 mg/L

Based on:

ThOD

Initial conc.:

26.7 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:

Test System Justification and Route of Administration:
The test system and route of administration was selected based on the OECD Guideline 301 (OECD, 1992), and in consideration of the physical/chemical properties of the test material. The test material occurs as a slightly viscous liquid and dissolves uniformly in water. Therefore test material was directly added (by weight) to the reaction vessels. The reference material is highly soluble in water, and volumetric portions were added to the reaction mixtures as a concentrated aqueous stock solution.

Solubility and Stability Assessment:
A prior determination of stability in the test medium was not relevant to biodegradation studies, as the test conditions were intended to promote degradation by biodegradation, hydrolysis, and oxidation/reduction reactions. The test material was expected to be soluble in water. The extent to which the test material was dissolved and dispersed in the test medium was assessed by analyzing dissolved organic carbon (DOC) in the biodegradation reaction mixtures at test initiation.

Chemicals and Reagents:
De-ionized water used to prepare the mineral medium and test chemical/reference substance stock solutions was purified though 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 pH of the finished mineral medium was recorded, and adjusted if necessary within the range of 7.2 – 7.6. The finished mineral medium was filter-sterilized with a Corning 0.2 μm membrane sterilization unit prior to addition of the inoculum. See Table 1 in "Other Methods" section for Mineral Composition.

Test Procedure:
The biodegradation reaction mixtures were prepared in specially designed 1-liter glass reaction vessels, each containing approximately 500 mL portion of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFE-coated magnetic stir bars. These vessels are also fitted with 20 x 105 mm glass side baffles to facilitate complete mixing/aeration of the stirred reaction mixtures. All reaction vessels were labeled using a numbering system for vessel identification. Inoculum Blanks, containing the inoculated mineral medium 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 dissolved organic carbon (DOC) concentrations in the absence of added test or reference material. Biodegradation of the reference material, aniline was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. These reaction mixtures contained approximately 100 mg/L aniline, which was added to the inoculated mineral medium as a concentrated aqueous solution. Biodegradation of the test material was determined in duplicate Test Mixtures by adding weighed portions of test material to the inoculated mineral medium (500 mL) at a concentration of 26.7 mg/L, yielding 48.3 mg/L ThOD.
After addition of test material and aniline to the appropriate vessels, the pH of the reaction mixtures were measured and adjusted as necessary to 7.4 ± 0.2, then stirred for approximately 30 minutes to homogenize their contents prior to initiation of the test. Samples (30 mL) of the Inoculum Blanks, Positive Controls, and Test Mixtures were collected for initial analyses of dissolved organic carbon (DOC) and nitrate/nitrite. Prior to measurement of initial oxygen and CO2 concentrations, the headspace volume of each individual reaction vessel was determined by the automated respirometer system. Other specific operating parameters for the respirometer system are described in detail in the Study File. The biodegradation reaction mixtures were incubated in the darkness at a temperature between 20 and 24°C, and maintained within ± 1°C. The reaction mixtures were continuously stirred by a PTFE-coated magnetic stir bar rotating at a setting of 150 r.p.m.

Frequency of Sampling:
Concentrations of oxygen and CO2 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 pH, DOC, nitrite, and nitrate concentrations in the Inoculum Blanks, Positive Controls, and Test Mixtures were determined.

Statistics and Calculations:
Descriptive statistics (mean, standard deviation) were used where applicable.

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

67.6

St. dev.:

1.9

Sampling time:

28 d

Remarks on result:

other: By the end of the 28-day test, the % biodegradation of the test material reached 67.6 ± 1.9% DO2 (mean ± 1 SD). Biodegradation was slightly slower than required to meet the 10-day window criterion.

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 illustrate the extent of biodegradation of the test material under the conditions of this test. The time required for average biodegradation to exceed 10% DO2 (i.e., the lag period) was 2.8 days and the 60% DO2 level was exceeded after 14.1 days (See Table 2 in "Any other information on results" section). By the end of the 28-day test, the % biodegradation of the test material reached 67.6 ± 1.9% DO2 (mean ± 1 SD). Therefore, the rate of test material biodegradation was slightly slower than required to meet the 10-day window criterion for determination of ready biodegradability in the manometric respirometry test.

Results with reference substance:

Biodegradation of the reference material (aniline) exceeded 60% by 4.6 days.

Biological Oxygen Demand (BOD):

By the end of the 28-day test, the % biodegradation of the test material reached 67.6 ± 1.9% DO2 (mean ± 1 SD).

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

Reaction Mixtures	Time (Days) to Achieve		DO2 (%)* at
	10% DO2	60% DO2	10-d Window	Day 28
Positive Controls	3.8	4.6	83.7 ± 1.2	91.7 ± 0.8
Test Mixtures	2.8	14.1	56.7 ± 0.3	67.6 ± 1.9

*mean ± standard deviation, n = 2

CO2 Evolution:

Two other OECD tests for ready biodegradability utilize measurements of CO2 evolution to indicate the extent of test material 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 measurements of CO2 evolution can substantiate the extent of test substance biodegradation and ready biodegradability conclusion derived from oxygen consumption. Biodegradation of the test material exceeded 10% DCO2 after 5.1 days, and after 28 days reached 47.9 ± 0.4% DCO2 (mean ± 1 SD). Therefore, the rates and extents of biodegradation determined from CO2 evolution confirm the

biodegradability of the test material under the conditions of this test.

DOC Analyses:

Analyses of DOC were performed on all vessels to determine the percent degradation of the test and reference materials. The analyses of the Test Mixtures at test initiation indicated a mean blank-corrected concentration of 15.2 mg/L, which based on a theoretical carbon content of 58.4%, equates to 26 mg/L of the test material. Therefore, the test material was considered as fully soluble at the nominal concentration tested (27 mg/L). The extent of DOC removal in the Test Mixtures was 92.1 ± 0.0 mg/L (mean ± 1 SD., n=2) at day 28. The mean blank-corrected DOC concentration in the Test Mixtures at day 28 were 1.20 mg/L. These results confirm the ultimate biodegradation of the test material and suggest that little or no persistent and water soluble degradation products were formed as a result of its biodegradation.

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 consumed an average of 55 mg/L oxygen over 28 days, where the OECD guideline indicates that this background oxygen consumption should not exceed 60 mg/L. The inoculum produced > 60% biodegradation of the reference material, aniline, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 4.6 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 91.7%, 59.1% and 92.4%, respectively, at the end of the test.

For the Test Mixtures 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 material biodegradation in the replicate Test Mixtures differed by < 6.6% DO2 over all sample intervals of the 28-day test. The maximum difference in percentage of aniline biodegradation in replicate Positive Control reaction mixtures differed by < 1.94% DO2 at the end of the 10-day window. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.

Temperature was recorded periodically throughout the study using a calibrated min/max digital thermometer. The recorded daily minimum temperatures averaged 21.6 ± 0.3°C (± 1 SD., n = 15) and the maximum temperatures averaged 22.0 ± 0.4°C, over the entire duration of this test.

The pH of the biodegradation reaction mixtures ranged from 7.08 to 7.33 and remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Mixtures decreased by no more than 0.24 pH units from their initial values over 28 days, and showed only a 0.07 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 materials evaluated in this test.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Biodegradation of the oxazolidinyl ethyl methacrylate substance exceeded the 60% pass criterion for demonstrating “ready biodegradability” in the manometric respirometry test. However, the rate of biodegradation was slightly slower than that required to meet the associated 10-day window criterion. The results of this test demonstrated that oxazolidinyl ethyl methacrylate is not inhibitory of the inoculum and can be classified as “readily biodegradable, but failing the 10-day window”, according to the Introduction to Section 3 (Degradation and Accumulation) of the OECD Guidelines (OECD, 2006).

Executive summary:

The ready biodegradability of oxazolidinyl ethyl methacrylate was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined mineral medium at a concentration of approximately 29.6 mg/L (dry solids). Biodegradation of the test material was evaluated in reaction mixtures at a concentration of approximately 26.7 mg/L, which was equivalent to approximately 48.3 mg/L theoretical oxygen demand (ThOD). Reaction mixtures were incubated in the darkness at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using an automated respirometer system. The onset of oxazolidinyl ethyl methacrylate biodegradation (i.e. oxygen consumption ≥ 10% of ThOD) occurred after 2.8 days in the Test Mixtures, and biodegradation exceeded the pass level of 60% ThOD consumption after 14.1 days. At the end of the 28 day test, the extent of biodegradation based on BOD, CO2 evolution and DOC removal reached 67.6 ± 1.9%, 47.9 ± 0.4%, and 92.1 ± 0.0% (mean ± 1 SD, n = 2), respectively. Thus, oxazolidinyl ethyl methacrylate is not inhibitory of the inoculum and can be classified as “readily biodegradable but failing the 10 -day window”. Other results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability test. These include parameters such as viability of the inoculum, control of pH and temperature, and precision in percentage biodegradation recorded among replicate test mixtures containing a biodegradable reference material. Biodegradation of the reference material (aniline) exceeded 60% by 4.6 days, verifying the viability of the activated sludge inoculum. Therefore, the results of this study are considered fully valid, and indicate that oxazolidinyl ethyl methacrylate exhibits potential for rapid and ultimate degradability in various aerobic environments.

Description of key information

Rapid biodegradation of Oxazolidinyl ethyl methacrylate (OXEMA) was observed in a ready biodegradation test according to test guideline OECD 301F, as it passed the criterion of 60% for demonstrating “ready biodegradability” in the manometric respirometry test. Nevertheless, the rate of biodegradation was slightly slower than that required to meet the associated 10-day window criterion. The results of this test demonstrated that oxazolidinyl ethyl methacrylate is not inhibitory of the inoculum and can be classified as “readily biodegradable, but failing the 10-day window”, according to the Introduction to Section 3 (Degradation and Accumulation) of the OECD Guidelines (OECD, 2006).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Additional information

In the key study, the ready biodegradability of OXEMA was determined using the OECD 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined mineral medium at a concentration of approximately 29.6 mg/L (dry solids). Biodegradation of the test material was evaluated in reaction mixtures at a concentration of approximately 26.7 mg/L, which was equivalent to approximately 48.3 mg/L theoretical oxygen demand (ThOD). Reaction mixtures were incubated in the darkness at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using an automated respirometer system. The onset of OXEMA biodegradation (i.e. oxygen consumption ≥ 10% of ThOD) occurred after 2.8 days in the Test Mixtures, and biodegradation exceeded the pass level of 60% ThOD consumption after 14.1 days. At the end of the 28 day test, the extent of biodegradation based on BOD, CO2 evolution and DOC removal reached 67.6 ± 1.9%, 47.9 ± 0.4%, and 92.1 ± 0.0% (mean ± 1 SD, n = 2), respectively. Thus, OXEMA is not inhibitory of the inoculum and can be classified as “readily biodegradable but failing the 10 -day window”.

In a supporting study, the inherent biodegradability of OXEMA was assessed by a Manometric Respirometry procedure (Barnes, 2002) based on OECD Procedure 302C (Inherent Biodegradability) and features of OECD Procedure 301F (Ready Biodegradability). Biodegradation of OXEMA achieved 15% of the ThODammonia (Theoretical Oxygen Demand without nitrification - 50.0 mg O₂) value after approximately 4 days of incubation, 60% by day 11 and up to 90.8% by day 28. Degradation had achieved>60% on day 13 for both ThODnitrite (Theoretical Oxygen Demand with nitrification - Nitrite)and ThODnitrate (Theoretical Oxygen Demand with nitrification – Nitrate) and up to 79.9% and 76.9% by the end of the test, respectively. Since degradation of OXEMA achieved 60% of the ThODnitrite and ThODnitrate, it is considered inherently or ultimately degradable.