4,4-dimethyloxazolidine

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:

September - October 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4-dimethyloxazolidine

EC Number:

257-048-2

EC Name:

4,4-dimethyloxazolidine

Cas Number:

51200-87-4

Molecular formula:

C5H11NO

IUPAC Name:

4,4-dimethyl-1,3-oxazolidine

Test material form:

liquid

Specific details on test material used for the study:

- Name: BIOBAN CS-1135 (4,4-dimethyl-oxazolidine)
- Source and lot/batch No.of test material: Dow Chemical Company, 1H25 DF87
- Purity: 66% by weight

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Activated sludge mixed liquor collected from the Midland Municipal Wastewater Treatment Plant (Midland, Michigan), > 90% from domestic sources.
- Preparation of inoculum for exposure: The sludge was pre-conditioned (aerated but not fed) to reduce the residual dissolved organic carbon. The mixed liquor suspended solids (MLSS) concentration of the activated sludge was determined to be 1960 ± 10 mg/L. Based on this determination, 16 liters of sterile mineral medium were inoculated with 245 mL of the mixed liquor to yield a final MLSS concentration of 30 mg/L. The pH of the inoculated mineral medium was 7.3.
- Pretreatment: The activated sludge was used after sampling from the treatment plant without adaptation.
- Concentration of sludge: 30 mg/L dry matter in the final mixture.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

>= 6 - <= 16 mg/L

Based on:

ThOD

Remarks:

20 to 50 mg/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
Stock solutions:
* mineral stock solution I (10mL/L): 8.5 g/L KH2PO4, 21.75 g/L K2HPO4, 50.4 g/L Na2HPO4.7H2O, 0.5 g/L NH4Cl, pH 7.4
* mineral stock solution II (1 mL/L): 36.4 g/L CaCl2.2H2O
* mineral stock solution III (1 mL/L): 22.5 g/L MgSO4.7H2O
* mineral stock solution IV (1 mL/L): 0.15 g/L FeCl3
* Final test medium: 10 mL of solution I and 1 mL of solutions II, III and IV per L of test medium

- Test temperature: 22 +-1°C
- pH: 7.4 +-0.2
- Continuous darkness: yes

TEST SYSTEM
- Test flasks: 1 L Pyrex-Bottle containing a total volume of test solution of 500 mL were incubated under continuous stirring at 150 r.p.m.
- The test and reference materials were added to the appropriate reaction mixtures as concentrated aqueous solutions to give 100 mg/L benzoate
- Tests performed with 2x 6.4 mg/L and 2x 16 mg/L test material
- Single biologically inhibited control reactions containing 6 to 16 mg/L of the test materials were prepared in chemically-sterilized mineral medium (250 mg/L HgCl2) to verify the absence of O2 consumption and/or CO2 evolution in the absence of biological activity
- Samples (20 mL) of the reaction mixtures in each vessel were removed to determine the initial concentrations of DOC, nitrite, and nitrate.
- After connection to the respirometer system, the reaction vessels were purged with ambient air, and the associated headspace volume of each individual reaction vessel was determined by the respirometer system.

CONTROL AND BLANK SYSTEM
- Inoculum blank (two replicates): none added
- Positive control (two replicates): 100 mg/L Sodium benzoate (200 mg ThOD/L)
- Toxicity control (one control for the tests with 6.4 mg/L and 16 mg/L test material): 100 mg/L Sodium benzoate (200 mg ThOD/L)
- Biologically inhibited control (one control for the tests with 6.4 mg/L and 16 mg/L test material): none added

SAMPLING
The test vessels were stirred by a stirring system for a maximum test period of 28 days. Measurements of gas phase O2 and CO2 in the reaction vessels occurred on 6-hour sample intervals over the entire 28-day test period. Upon completion of day 28 gas measurements, the reaction mixtures in each vessel were sampled for final DOC, nitrite, and nitrate measurements. The pH of each reaction mixture was also determined at the end of the 28-day test.

ANALYTICAL METHODS:
Gas phase measurements of O2 and CO2 were performed using the Columbus MicroOxymax® system. The system used a paramagnetic oxygen sensor with a measurement range of 19.0 to 21.0% (vol.) O2, and a non-dispersive infrared (NDIR) CO2 detector with a measurement range of 0 to 0.8% (vol.). Oxygen and CO2 measurements were normalized to a pressure of 800-mm Hg to eliminate effects of fluctuating atmospheric pressure.

DOC concentrations in aqueous samples were determined using a Shimadzu model TOC-5000 analyzer equipped with an ASI-5000 autosampler.
Prior to analysis, samples (20 mL) from the reaction mixtures were filtered through 0.45-µm nylon syringe filters that had been flushed with approximately 10 mL MilliQ® water. The concentrations of nitrite and nitrate in the filtered reaction solutions were determined by ion chromatography using a Dionex Model DX-120 ion chromatograph.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

94.9

Sampling time:

28 d

Remarks on result:

other: The test material concentration of 16mg/L (equivalent to approximately 50 mg/L ThOD) fell within the standard guidelines of the test, and the results from these reaction mixtures were used.

Details on results:

The time required for biodegradation of 16 mg/L of the test material to exceed 10% DO2 (i.e., the lag period) was 1.9 days and a total of 10.4 days were required for biodegradation (DO2) to exceed 60%. Since biodegradation exceeded the 60% level within 10 days of the onset of biodegradation, the test material can be classified as “readily biodegradable” according to the OECD 301F guideline. Biodegradation reached 94.9% DO2 at 28 days.
Comparable results were observed in reaction mixtures containing 6-mg/L test material: Biodegradation reached 10% DO2 in 0.6 days, 60% DO2 in 9.4 days, and 93.0% DO2 after 28 days.

Mineralization results for the test and reference compounds were consistent with the biodegradation results based on oxygen consumption. However, the calibration check of the CO2 sensor in the respirometer system at the conclusion of the study did not meet acceptance criteria. Therefore, biodegradation results based on mineralization are not reported.

The extent of biodegradation of the test material determined by respirometry was consistent with the extent of removal of dissolved organic carbon (DOC) in the reaction mixtures. Degradation based on DOC removal ranged from 95 to 98% after 28 days for 6 to 16 mg/L of test material.

Any other information on results incl. tables

Test Validation

- The inoculum used in this test produced > 60% biodegradation of the positive control, benzoate, within the required 10-day window prior to day 14 of the test.

Biodegradation of benzoate based on O2 consumption averaged 90.7% after 28 days. Biodegradation of benzoate based on DOC removal reached 99%. Levels of oxygen consumption in the inoculum blanks remained well below the maximum allowed level of 60 mg/L. Oxygen consumption in the inoculum blank reactions reached a maximum level of 24.7 mg/L after 28 days.

- The maximum observed difference in DO2 values among replicate reaction mixtures containing 16 mg/L test material was 3.3% for reaction mixtures containing 16 mg/L test material and 2.0% for

reaction mixtures containing benzoate. These values are within the validation criteria.

- The average of the recorded temperatures was 22.6 ± 0.3 °C. This average incubation temperature falls within the required range of 22 ± 2 °C.

- The pH of the biodegradation reaction solutions showed minimal variation over the course of the study, and remained within the required range of 6 - 8.5 after 28 days. After 28 days, the difference in pH values between the reaction mixtures containing the test material and the inoculum blanks did not exceed 0.4 pH units.

Toxicity Controls

Biodegradation measured in the toxicity control reactions containing 100 mg/L benzoate and 6 to 16 mg/L test material indicated that the test material was not inhibitory to the microbial inoculum according to the OECD 301 F guidelines. Net oxygen consumption in the toxicity control reaction exceeded 85% of the total added ThOD from benzoate and test material after 14 days. The OECD specifies that net oxygen consumption equivalent to less than 25% of the total applied ThOD within 14 days is indicative of inhibition by the test material. Therefore, there is no evidence for inhibition of the inoculum by the test material under the conditions of the test.

Biologically Inhibited Controls

Biologically inhibited control reaction mixtures were prepared with 8 to 20 mg/L test materials in inoculated mineral medium containing 250 mg/L HgCl2. These reaction mixtures showed no cumulative consumption of O2 or production CO2 over the 28-day duration of the test. Therefore, it can be concluded that the O2 consumption and CO2 production observed in the viable test reaction mixtures was due solely to biological activity.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

A 28-d ready biodegradability test (OECD 301F, Manometric Respirometry Test) using activated sludge of a municipal sewage treatment plant indicated that 4,4-dimethyl-oxazolidine (CAS 51200-87-4) reached a biodegradation of 94.9% based on O2 consumption. The substance reached the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window and, therefore, can be termed as readily biodegradable.

Executive summary:

The ready biodegradability of the test item (CAS no. 51200 -87 -4) was evaluated using the OECD Guideline 301 F: Manometric Respirometry Test. Biodegradation of the compound was determined at an initial concentration of 16 mg/L in reaction mixtures containing a dilute municipal activated sludge inoculum. Oxygen consumption resulting from biodegradation of the compound was measured over 28 days using a Columbus Instruments MicroOxymax® respirometer system (Columbus Instruments, Inc., Columbus, Ohio).

The results of this test demonstrate that the test material meets current OECD criteria for “ready biodegradability” in the Manometric Respirometry Test. Biodegradation as determined from oxygen consumption compared to the theoretical oxygen demand (ThOD) of the compound surpassed the required 60% level within a 10-day period following the onset of biodegradation. A lag period of 1.9 days was required before greater than 10% biodegradation occurred, and 10.4 total days were required for biodegradation to exceed the 60% level. Net oxygen consumption after 28 days was equivalent to 94.9% biodegradation of the test material.

The suitability of the test procedure and microbial inoculum was verified by rapid biodegradation of a positive control compound (sodium benzoate) in the reaction mixtures. Biodegradation of benzoate based on O2 consumption exceeded 60% after only 3.3 days, and reached 90.7% after 28 days. Toxicity control reactions containing a mixture of 100 mg/L benzoate and 16 mg/L test material showed no evidence for inhibition of biodegradation. The observed O2 consumption in the biodegradation reactions can be attributed solely to biological activity, as no net O2 consumption was observed in biologically inhibited control reactions containing 16 mg/L of the test material.