3,4-dihydro-2H-1,4-benzoxazin-6-ol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2021-02-04 to 2021-06-29

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

yes

Remarks:

Temperature range was 19.0 – 22.7 °C instead of 20.0 ± 1.0 °C. As degradation of the positive control was in the normal range this is considered as uncritical concerning the outcome of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.29 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test))

Deviations:

yes

Remarks:

Temperature range was 19.0 – 22.7 °C instead of 20.0 ± 1.0 °C. As degradation of the positive control was in the normal range this is considered as uncritical concerning the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from a biologic sewage treatment plant was used. The chosen plant was at Maxéville WWTP, 54320 France. The chosen plant treats mostly domestic sewage. The sludge was taken from the activation basin, one day before start of the test.
Date of collection: 10. Feb. 2021, batch no: E20210210

- Pretreatment: The sludge was filtrated through a cloth, washed with test medium 2x. The solids were then resuspended in test medium and aerated. The dry matter was determined with 3.22 g suspended solids / L.
- Concentration of sludge: 15 mg suspended solids/L

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Initial conc.:

16.15 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: All chemicals used in the test were “analytical grade” or >97.5%. Composition: Solution a 10 mL, Solution b 1 mL, Solution c 1 mL, Solution d 1 mL, H2O demin. ad 1000 mL. Composition of solutions a-d as in the guideline.

- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: room temperature (19.0 – 22.7 °C)
- pH: not specified
- pH adjusted: no
- CEC (meq/100 g): not specified
- Aeration of dilution water: not specified
- Suspended solids concentration: 15 mg/L
- Continuous darkness: The test was performed without direct lighting

TEST SYSTEM
- Culturing apparatus: The test was conducted with a volume of 150 mL in each test vessel. All glassware was cleaned with the laboratory cleaning agent and then rinsed with tap water (3x), diluted HCL (1x), tap water (3x) and diluted water (3x). 250 mL-narrow-neck-flasks were used as test vessels. These were closed with lids and silicon septa. In the lid is a small hole, through which the NaOH 7 m was dosed using a needle.
- Number of culture flasks/concentration: Two blank controls, 2 flasks containing aniline as positive control, 2 flasks containing test item, one flask containing test item and 0.05 g HgCl2/L, but no inoculum (testing for abiotic degradation) and one flask containing test item and aniline (testing for toxic effects) were tested at each point of measurement. Additionally, one blank control was analysed in order to determine IC stemming from medium and/or NaOH.
- Measuring equipment: Analyses of the emitted CO2 were made by inorganic carbon (IC) measurement (Carbon analyser TOC multi N/C 2100S, Analytik Jena)
- Test performed in open system: The test was performed in sealed bottles with a headspace of air, which provides a reservoir of oxygen for aerobic biodegradation.
- Details of trap for CO2 and volatile organics if used: To each vessel to be measured, 1.5 mL of NaOH 7 m was added by syringe and needle and the vessel was shaken for 1 hour again. After settling down, a sample of 1.5 mL was pipetted in a TOC-vials which was closed immediately. Each vial was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions (containing a mixture of Na2CO3 and NaHCO3). Quality control samples were measured once on each measuring day. At each sampling date, a blank containing mineral medium and NaOH 7 m was measured and the IC of the blank was subtracted from the IC of all sampled vessels.


SAMPLING
- Sampling frequency: the created CO2 in the vessels was determined on days 0, 4, 7, 11, 14, 19, 22, 25, 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, see above
- Abiotic sterile control: yes, see above
- Toxicity control: yes, see above

Reference substance:

aniline

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

28 d

Results with reference substance:

8.4% degradation in 4d, 68.5% in 7d and 74.3% in 14d.

Degradation values in %:

Values on day 28 were calculated from the means of the triplicate measurement.

Day	Test (mean of 2 flasks)	Abiotic control	Toxicity Control
4	-2.3	-13.9	32.4
7	2.7	17.9	40.5
11	2.7	-20.3	45.9
14	-1.2	-18.4	34.2
19	2.9	-18.5	47.5
22	1.7	-20.5	37.2
28	5.7	-19.2	44.6

 

Validity criteria fulfilled:

yes

Remarks:

Degradation of positive control: 60% within 7d Mean IC content of controls at the end of the test: 3.62 mg/L. Should be below 3, but The mean IC value > 3 mg/L is due to the fact, that a higher amount of sludge was added. This can be stated as uncritical.

Interpretation of results:

not readily biodegradable

Conclusions:

Degradation reached 6 % at the end of the test duration. The 10-day -window was not detected. Therefore, the test item can be considered as “not readily biodegradable”.

Executive summary:

The objective of the study was to assess the ready biodegradation potential of the test item according to the OECD 310 guideline and the EU C29 method and following GLP. The test item was tested using a concentration of 16.15 mg/L in test medium following ISO 14593:2005. Aniline was chosen as positive control
Activated sludge from a sewage treating plant was used as inoculum (concentration 15 mg suspended solids/L). The test was left running for 28 days.
All validity criteria were met. Degradation of the positive control was 69 % after 7 days.

The following data were determined for the test item 
10-day-window: not detectable
degradation at the end of 10-day-window none
degradation at the end of the test 6 %
Therefore, the test item is not readily biodegradable following OECD 310 and not ultimately biodegradable, within 28 days.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-01-07 to 2020-02-27

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:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Conditioning/Preparation of inoculum for exposure: The aerobic activated sludge used for this study was deposited for 30 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated until use. This suspension was used for the experiment.
- Concentration of sludge: final sludge concentration in test flasks: 28.7 mg sludge/L

Duration of test (contact time):

28 d

Initial conc.:

103.1 mg/L

Based on:

test mat.

Initial conc.:

185.4 mg/L

Based on:

ThOD

Remarks:

ThODNH4

Initial conc.:

229.1 mg/L

Based on:

ThOD

Remarks:

ThODN03

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Reconstituted Test Water: Analytical grade salts were added to ultrapure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; the pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with ultrapure water. The pH was 7.6.
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 19°C
- pH: 7.6 (measured at the start of the test); 7.6 to 7.7 (measured at the end of the test)
- pH adjusted: not specified
- CEC (meq/100 g): not specified
- Aeration of dilution water: no
- Suspended solids concentration: final sludge concentration in test flasks: 28.7 mg sludge/L
- Continuous darkness: yes
- Other: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System).
- Test performed in closed vessels : yes
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

SAMPLING
- Sampling/measurement frequency: each day from day 1 to day 28
- Sterility check if applicable: not specified
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 flasks; The inoculum control was also used for other studies which ran in parallel
- Abiotic sterile control: 1 flask, poisoned with CuSO4 (stock solution of 1 g/L)
- Toxicity control: 1 flask
- Procedure control: 1 flask; The procedure control was also used for other studies which ran in parallel

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

11

Sampling time:

28 d

Remarks on result:

other: Mean biodegradation percentage based on ThODNO3

Key result

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

28 d

Remarks on result:

other: Mean biodegradation percentage based on ThODNH4

Details on results:

The 10-day windows began on day 19 after application, the mean value was calculated to be 10% biodegradation (ThODNO3) and on day 15 (ThODNH4). Therefore, 10-day window could not be completed as the test was terminated after 28 days. The mean biodegradation percentage based on ThODNO3 at the end of the test at day 28 was 11%; the criterion of the 10 day window was not met.
If no nitrification is considered, the mean biodegradation was 14% (ThODNH4) after 28 days of incubation. The criterion of the 10 day window was not fulfilled.

Validity criteria:
Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 was 5% (ThODNH4) and 4% (ThODNO3). The validity criterion was fulfilled.
Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 15 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
pH-Value: The pH-value of the test item flasks at the end of the test was 7.6 and 7.7 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
Toxicity Control: If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on ThODNH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 40% at day 14; the test item was not inhibitory. The biodegradation based on ThODNO3 was 36%.

The oxygen demand in the abiotic control was 30 mg/L during the test duration. At the end of the test after 28 days, the biodegradation in the abiotic control was 16% (based on ThODNH4) and 13% (based on ThODNO3).

Results with reference substance:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was sufficiently degraded to 68% after 14 days and to 74% after 28 days of incubation.

Validity criteria fulfilled:

yes

Remarks:

See above

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of the test item never reached 60%. Therefore, the test item is considered to be not readily biodegradable.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.
According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Executive summary:

The aim of the study was to determine the ready biodegradability of the test item according to OECD 301F guideline and C.4-D method (manometric respirometry test) over 28 days and following GLP.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The test Item loading rate was 103.1 mg/L corresponding to an oxygen demand of about 185.4 mg/L (ThODNH4) and 229.1 mg/L (ThODN03).

The mean biodegradation of 10% of the test item was reached at day 15 (ThODNH4) and at day 19 (ThODNO3). At the end of the 10-day window at day 25, the mean degradation of the test item was 14% (ThODNH4) and therefore the 10 day window criterion was not met. The mean biodegradation at test end after 28 days was 14% (ThODNH4) and 11% (ThODNO3).

Therefore, the test item is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.

The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

Two tests were performed :

The first test was performed according to OECD 301F guideline and C.4-D method (manometric respirometry test) over 28 days and following GLP. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The test Item loading rate was 103.1 mg/L corresponding to an oxygen demand of about 185.4 mg/L (ThODNH4) and 229.1 mg/L (ThODN03). The mean biodegradation of 10% of the test item was reached at day 15 (ThODNH4) and at day 19 (ThODNO3). At the end of the 10-day window at day 25, the mean degradation of the test item was 14% (ThODNH4) and therefore the 10 day window criterion was not met. The mean biodegradation at test end after 28 days was 14% (ThODNH4) and 11% (ThODNO3). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum. According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

The second was conducted according to the OECD 310 guideline and the EU C29 method and following GLP. The test item was tested using a concentration of 16.15 mg/L in test medium following ISO 14593:2005. Aniline was chosen as positive control. Activated sludge from a sewage treating plant was used as inoculum (concentration 15 mg suspended solids/L). The test was left running for 28 days.
All validity criteria were met. Degradation of the positive control was 69 % after 7 days. The following data were determined for the test item :
10-day-window: not detectable
degradation at the end of 10-day-window none
degradation at the end of the test 6 %
Therefore, the test item is not readily biodegradable following OECD 310 and not ultimately biodegradable, within 28 days.