(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

Adopted July 17, 1992

Deviations:

yes

Remarks:

On nominal day 27 the aeration of blank bottle B was broken down due to a clogged air filter. Aeration was restored by replacing the filter. Evaluation: Such a short (<1 day) breakdown in aeration has no influence on the outcome of the test.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

Deviations:

yes

Remarks:

On nominal day 27 the aeration of blank bottle B was broken down due to a clogged air filter. Aeration was restored by replacing the filter. Evaluation: Such a short (<1 day) breakdown in aeration has no influence on the outcome of the test.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

EC Number:

605-404-6

Cas Number:

16535-55-0

Molecular formula:

C11 H9 N O6

IUPAC Name:

(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

Test material form:

solid: particulate/powder

Details on test material:

Off-white crystalline powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Laboratory culture: /
- Method of cultivation: The freshly obtained sludge was used immediately.
- Storage conditions: /
- Storage length: /
- Preparation of inoculum for exposure: /
- Pretreatment: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge.
- Initial cell/biomass concentration: /
- Water filtered: yes
- Type and size of filter used, if any: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

46.63 mg/L

Based on:

test mat.

Remarks:

Test bottle A

Initial test substance concentration 2

Initial conc.:

46.68 mg/L

Based on:

test mat.

Remarks:

test bottle B

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
- Additional substrate: /
- Solubilising agent (type and concentration if used): /
- Test temperature: between 22.2 and 23.3°C.
- pH: 7.6-7.8
- pH adjusted: no
- CEC (meq/100 g): /
- Aeration of dilution water: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Suspended solids concentration: 3.6 g/L
- Continuous darkness: yes
- Other: /

TEST SYSTEM
- Culturing apparatus: /
- Number of culture flasks/concentration:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Method used to create anaerobic conditions: /
- Measuring equipment: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
- Test performed in closed vessels due to significant volatility of test substance: /
- Test performed in open system: /
- Details of trap for CO2 and volatile organics if used: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
- Other: /

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On the penultimate day, the pH of all test media was measured and 1 mL of concentrated HCl (37%, Merck) was added to the test bottles. Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).
- Sterility check if applicable: /
- Sample storage before analysis: /
- Other: /

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum (2 bottles)
- Abiotic sterile control: /
- Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Other: Positive control: containing reference item and inoculum (1 bottle).

STATISTICAL METHODS:

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

final concentration of 40 mg sodium acetate per litre (12 mg TOC/L)

Results and discussion

Test performance:

In the toxicity control, more than 25% biodegradation occurred within 14 days (73%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate (i.e. procedure control), which showed a normal biodegradation curve

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottle A

Value:

84

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottle B

Value:

96

Sampling time:

28 d

Details on results:

Relative biodegradation values calculated from measurements performed during the test period revealed 84% and 96% biodegradation of CH02672, for vessel A and B, respectively (based on ThCO2).
However, average biodegradation of CH02672 in vessel A and B did not reach ≥60% within a 10-day window. Thus, the criterion for ready biodegradability was not met.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

In conclusion, CH02672 was not readily biodegradable under the conditions of the modified Sturm test presently performed. Since results of the present test indicate that the pass level criterion was fulfilled but the 10-day window criterion was not fulfilled, the present test can be used to indicate inherent biodegradability.1

Executive summary:

The objective of the study was to evaluate test item CH02672 for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

Procedures described in this report were in compliance with OECD guideline No. 301 B, 1992. In addition, procedures were designed to meet test methods of ISO standard 10634, 1995.

CH02672 was an off-white crystalline powder with a purity of >99.9% (HPLC). The test item was tested in duplicate at a target concentration of 23 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO2production (ThCO2) of CH02672 was calculated to be 1.93 mg CO2/mg.

The study consisted of six bottles:

•   2 inoculum blanks (no test item),

•   2 test bottles (CH02672),

•   1 procedure control (sodium acetate) and

•   1 toxicity control (CH02672 plus sodium acetate).

Since CH02672 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15). Relative biodegradation values calculated from measurements performed during the test period (28 days) revealed 84% and 96% biodegradation of CH02672, for vessel A and B, respectively (based on ThCO2). However, biodegradation did not exceed 60% within 10 days of achieving 10%. In the toxicity control, CH02672 was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, CH02672 was not readily biodegradable under the conditions of the modified Sturm test presently performed. Since results of the present test indicate that the pass level criterion was fulfilled but the 10-day window criterion was not fulfilled, the present test can be used to indicate inherent biodegradability.