p-benzoquinone dioxime

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:

30-01-2018 to 07-06-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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, (1995)

Deviations:

no

GLP compliance:

yes

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): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Laboratory culture: Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
- Method of cultivation: See above.
- Storage conditions: See above.
- Storage length: < 1 week.
- Preparation of inoculum for exposure: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water. The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Pretreatment: 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
- Concentration of sludge: After treatment the concentration of suspended solids (SS) was determined to be 2.9 g/L in the concentrated sludge as used for experiment 1. The magnetically stirred sludge was used as inoculum at the amount of 4 mL per litre of mineral medium, leading to a SS concentration of 12 mg/L. For experiment 2 the concentration of SS in the sludge was determined to be 3.4 g/L. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 10 mg/L
- Initial cell/biomass concentration: See above.
- 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 conc.:

23 mg/L

Based on:

test mat.

Remarks:

Experiment 1

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

Experiment 1

Initial conc.:

10 mg/L

Based on:

test mat.

Remarks:

Experiment 3

Initial conc.:

5 mg/L

Based on:

TOC

Remarks:

Experiment 3

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. The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Solution 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
Solution B): 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
Solution C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
Solution D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
- Additional substrate: Not applicable.
- Solubilising agent (type and concentration if used): Not applicable.
- Test temperature: 22 ±1 °C
- pH: In experiment 1: 7.5 to 7.6 (test item vessels), 7.6 to 7.8 (procedure control), 7.6 (blank control), 7.5 to 7.9 (toxicity control)
In experiment 2: - (test item vessels), 7.6 (procedure control), 7.6 (blank control), 7.6 (toxicity control)
In experiment 3: 7.6 (test item vessels), 7.6 (procedure control), 7.6 (blank control), 7.5 (toxicity control)
- pH adjusted: Yes. In experiment 2: blank control, procedure control and toxicity control only. 7.7 to 7.6/7.5 at start of test (only). Adjusted using 1 M HCl
In experiment 3: Test item, blank control, procedure control and toxicity control only. 7.8/7.7 to 7.6/7.5 at start of test (only). Adjusted using 1 M HCl
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: After treatment the concentration of suspended solids (SS) was determined to be 2.9 g/L in the concentrated sludge as used for experiment 1. The magnetically stirred sludge was used as inoculum at the amount of 4 mL per litre of mineral medium, leading to a SS concentration of 12 mg/L. For experiment 2 the concentration of SS in the sludge was determined to be 3.4 g/L. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 10 mg/L
- Continuous darkness: Yes. Brown coloured glass bottles were utilised. Media was excluded from light.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: Duplicate.
- Method used to create aerobic conditions: synthetic air was passed 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: Not applicable.
- 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). Three 100 mL bottles contained in series.
- Test performed in closed vessels due to significant volatility of test substance: Not applicable.
- Test performed in open system: synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Details of trap for CO2 and volatile organics if used: See measuring equipment.
- Other: Not applicable.

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, with the exception of experiment 2 where the blanks were titrated until day 14. Titrations for the procedure and toxicity control were made over a period of at least 14 days.
- Sampling method: Titration.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not applicable.
- Other: Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2 litre test bottles containing medium with microbial organisms and mineral components.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: Not applicable.
- Toxicity control: Yes.
- Other: Procedure control (reference item: sodium acetate and inoculum only).

Reference substance:

acetic acid, sodium salt

Remarks:

40 mg/L

Test performance:

1. The reference item was biodegraded by at least 60% within 14 days. (Actual: 88%, 86% and 82% for experiments 1, 2 and 3 respectively).
2. The difference of duplicate values for %-degradation of the test item was always less than 20%. (Actual: < 1% and < 5% in experiments 1 and 3)
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L. (Actual: 31.5 mg/L and/or 37.1 mg/L in experiments 1 and 3)
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water, carbon levels < 500 ppb), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L)

Parameter:

% degradation (CO2 evolution)

Remarks:

mean (n=2)

Value:

2

Sampling time:

28 d

Remarks on result:

other: Experiment 1 (2% and 1% individually)

Parameter:

% degradation (CO2 evolution)

Remarks:

mean (n=2)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Experiment 2 (toxicity trial) due to inhibition seen in Experiment 1 (no definitive testing of test item alone)

Parameter:

% degradation (CO2 evolution)

Value:

5

Sampling time:

28 d

Remarks on result:

other: Experiment 3 (3% and 7% individually)

Details on results:

In experiment 1: In the toxicity control, less than 25% biodegradation occurred within 14 days (20%, based on ThCO2). Therefore, the test item was assumed to inhibit microbial activity at a target concentration of 23 mg/L.
In experiment 3: In the toxicity control, more than 25% biodegradation occurred within 14 days (58%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity at a target concentration of 10 mg/L.

Results with reference substance:

Sodium Benzoate attained 88%, 86% and 82% degradation after 14 days in experiments 1, 2 and 3 respectively, thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mean biodegradation for two independent experiments (each in duplicate) was 2 % and 5% at day 28, respectively.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301B guideline under GLP using. The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage. The organic carbon content was based on the molecular formula of the test item. The Theoretical CO2 production (ThCO2) of the test item was calculated to be 1.91 mg CO2/mg. The test item was tested in duplicate at a target concentration of 23 mg/L, corresponding to 12 mg TOC/L. The toxicity control showed a possible inhibitory effect in experiment 1. A second full test was performed at a target concentration of 10 mg/L, corresponding to 5 mg TOC/L. The results of the second full test support the findings of the first experiment. The test included two inoculum blanks (no test item), two test item bottles, one procedure control (sodium acetate) and a toxicity control (test item plus sodium acetate). Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 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 the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the procedure and toxicity control (last CO2 measurement on day 15). The relative biodegradation values calculated from the measurements performed during experiment 1 revealed no biologically relevant biodegradation of test item (2% and 1%, based on ThCO2) at a target concentration of 23 mg/L. The relative biodegradation values calculated from the measurements performed during experiment 3 revealed no biologically relevant biodegradation of test item (7% and 3%, based on ThCO2) at a target concentration of 10 mg/L. In the toxicity control, test item was found to possibly inhibit microbial activity at the initially tested target concentration of 23 mg/L. No inhibitory effects were observed at a target concentration of 10 mg/L. All acceptability criteria were met. Under the conditions of the study, the substance is not readily biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 2 - 5 % (28 -days), OECD TG 301B, 2018

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Key Study : OECD TG 301B, 2018 - The ready biodegradability test was carried out according to OECD TG 301B guideline under GLP using. The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage. The organic carbon content was based on the molecular formula of the test item. The Theoretical CO2 production (ThCO2) of the test item was calculated to be 1.91 mg CO2/mg. The test item was tested in duplicate at a target concentration of 23 mg/L, corresponding to 12 mg TOC/L. The toxicity control showed a possible inhibitory effect in experiment 1. A second full test was performed at a target concentration of 10 mg/L, corresponding to 5 mg TOC/L. The results of the second full test support the findings of the first experiment. The test included two inoculum blanks (no test item), two test item bottles, one procedure control (sodium acetate) and a toxicity control (test item plus sodium acetate). Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 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 the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the procedure and toxicity control (last CO2 measurement on day 15). The relative biodegradation values calculated from the measurements performed during experiment 1 revealed no biologically relevant biodegradation of test item (2% and 1%, based on ThCO2) at a target concentration of 23 mg/L. The relative biodegradation values calculated from the measurements performed during experiment 3 revealed no biologically relevant biodegradation of test item (7% and 3%, based on ThCO2) at a target concentration of 10 mg/L. In the toxicity control, test item was found to possibly inhibit microbial activity at the initially tested target concentration of 23 mg/L. No inhibitory effects were observed at a target concentration of 10 mg/L. All acceptability criteria were met. Under the conditions of the study, the substance is not readily biodegradable.