6-amino-2-(2,4-dimethylphenyl)-1H-benz[de]isoquinoline-1,3(2H)-dione

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:

28 November 2016 to 02 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test

Version / remarks:

1999

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"

Version / remarks:

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: 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.
- Preparation of inoculum for exposure: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was allowed to settle (32 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: The concentration of suspended solids was determined to be 5 g/L in the concentrated sludge.

Duration of test (contact time):

28 d

Initial conc.:

16 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

Based on the molecular formula

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Stock solutions of mineral components. 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
Medium 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.
Medium B: 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
Medium C: 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
Medium D: 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
Pre-incubation medium: 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.
- Test temperature: The temperature recorded in a vessel with water in the same room varied between 21.8 and 23.6 °C.
- pH: Ranged from 7.6 - 8.2 during the test.
- pH adjusted: Yes, adjusted to 7.6 using 1 M HCl at the start of the test.
- Aeration of dilution water: Yes, during the test period the test media were aerated continuously.
- Suspended solids concentration: The concentration of suspended solids was determined to be 5 g/L in the concentrated sludge. Before use, the sludge was allowed to settle and the supernatant liquid was used as inoculum.
- Continuous darkness: Yes.
- Other: Test solutions: Since the test material 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 (test material bottle A: 32.6 mg; test material bottle B: 32.5 mg and toxicity control bottle: 32.1 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test material. 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 material and the test organisms. Furthermore, the test medium was daily swirled around, since the test material tended to float on the water surface. A correction factor for purity or composition was not applied. Preparation was as much as possible performed under low light conditions.
- Other: Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
- Preparation: At the start of the test (day 0), test and reference material 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.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles.
- Number of culture flasks/concentration: 2 bottles.
- Method used to create aerobic conditions: Synthetic air (CO2 < 1 ppm). 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 to 100 mL/min).
- Details of trap for CO2 and volatile organics if used: 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. Barium hydroxide 0.0125 M Ba(OH)2 and freshly prepared 0.0125 M Ba(OH)2 solution were both stored in a sealed vessel to prevent absorption of CO2 from the air.

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 suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: 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 standardised HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule)).
Each time the CO2-absorber nearest to the test bottle was removed for titration, each of the remaining two absorbers was 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) was used as pH-indicator.
On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37 %) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
- pH measurement: At the start of the test (day 0) and on day 28, before addition of concentrated HCl.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles.
- Toxicity control: 1 bottle (toxicity control containing test material, reference material and inoculum).
- Positive control containing reference material and inoculum: 1 bottle. A solution of sodium acetate was prepared by dissolving 400.7 mg in Milli-RO water and making this up to a total volume of 100 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).

STATISTICAL METHODS:
The theoretical CO2 production was calculated from the molecular formula.
ThCO2, expressed as mg CO2/mg test material, was calculated as follows:
ThCO2 = (No. of carbon atoms in test material × Molecular weight CO2) / Molecular weight test material

The first step in calculating the amount of CO2 produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2 produced by a test material is determined by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps.

The amount of 0.05 N HCl titrated is converted into mg of CO2 produced:
mg CO2 = ((0.05 × Δ mL HCl titrated) / 2) x 44 = 1.1 × Δ mL HCl titrated

Relative biodegradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production, based on the total carbon content of the amount of test material present in the test bottles. A figure of more than 10 % biodegradation was considered significant.
The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. If applicable, the number of days is calculated from the attainment of 10 % biodegradation until 60 % biodegradation. Should this period be ≤ 10 days (10-day window), then the test material is designated as readily biodegradable.

Toxicity control: if less than 25 % biodegradation (based on ThCO2 of the test and positive control materials combined) occurred within 14 days, the test material was assumed to be inhibitory.

The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in mL of titrant) between the blank Ba(OH)2 traps and untreated Ba(OH)2 (background).

Reference substance:

acetic acid, sodium salt

Test performance:

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 9 - <= 14

Sampling time:

28 d

Remarks on result:

other: Standard deviation not reported.

Details on results:

The ThCO2 of the test material was calculated to be 2.78 mg CO2/mg.

The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 9 % biodegradation of the test material (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25 % biodegradation occurred within 14 days (36 %, based on ThCO2). Therefore, the test material was assumed not to inhibit microbial activity.

Acceptability of the test
1. The positive control material was biodegraded by at least 60 % (76 %) within 14 days.
2. The difference of duplicate values for %-degradation of the test material was always less than 20 (≤ 6 %).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (42.4 mg CO2 per 2 litres of medium, corresponding to 21.2 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test material (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 material, 12 mg TOC/L).
 
Since all criteria for acceptability of the test were met, this study was considered to be valid.

Results with reference substance:

The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg. Functioning of the test system was checked by testing the reference material sodium acetate, which showed a normal biodegradation curve. The positive control exhibited 76 % degradation within 14 days.

Table 1: CO2 production and percentage biodegradation of the test material (bottle A)

Day	HCl (0.05 N) Titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation* (%)
	Blank (mean)	Bottle A
2	48.54	47.53	1.01	1.1	1.1	1
5	46.22	45.00	1.22	1.3	2.5	3
7	46.43	45.72	0.71	0.8	3.2	4
9	46.61	45.79	0.81	0.9	4.1	5
14	46.30	43.82	2.48	2.7	6.9	8
19	44.55	42.26	2.29	2.5	9.4	10
23	43.99	42.55	1.44	1.6	11.0	12
27	45.73	44.93	0.80	0.9	11.8	13
29	47.07	46.02	1.04	1.1	13.0	14
29	47.84	47.77	0.06	0.1	13.1	14
29	49.16	49.15	0.01	0.0	13.1	14

*Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test material: 90.6 mg CO2/2 L

Table 2: CO2 production and percentage biodegradation of the test material (bottle B)

Day	HCl (0.05 N) Titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation* (%)
	Blank (mean)	Bottle B
2	48.54	46.78	1.76	1.9	1.9	2
5	46.22	45.96	0.26	0.3	2.2	2
7	46.43	46.34	0.09	0.1	2.3	3
9	46.61	46.21	0.39	0.4	2.8	3
14	46.30	45.16	1.14	1.3	4.0	4
19	44.55	43.56	0.99	1.1	5.1	6
23	43.99	43.11	0.88	1.0	6.1	7
27	45.73	45.11	0.62	0.7	6.7	7
29	47.07	46.18	0.88	1.0	7.7	9
29	47.84	47.51	0.32	0.4	8.1	9
29	49.16	49.12	0.04	0.0	8.1	9

*Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test material: 90.4 mg CO2/2 L

Table 3: CO2 production and percentage biodegradation of the positive control material

Day	HCl (0.05 N) Titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation* (%)
	Blank (mean)	Positive control
2	48.55	41.03	7.51	8.3	8.3	10
5	46.22	26.71	19.51	21.5	29.7	35
7	46.43	34.68	11.75	12.9	42.6	50
9	46.61	38.51	8.10	8.9	51.6	60
14	46.30	34.20	12.10	13.3	64.9	76

*Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.7 mg CO2/2 L

Table 4: CO2 production and percentage biodegradation of the toxicity control

Day	HCl (0.05 N) Titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation* (%)
	Blank (mean)	Toxicity control
2	48.54	39.23	9.31	10.2	10.2	6
5	46.22	24.36	21.86	24.0	34.3	20
7	46.43	32.86	13.57	14.9	49.2	28
9	46.61	41.21	5.40	5.9	55.1	32
14	46.30	38.93	7.37	8.1	63.3	36

*Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test material and positive control: 175.0 mg CO2/2 L (ThCO2 test material: 89.2 mg CO2/2 L + ThCO2 sodium acetate: 85.7 mg CO2/2 L)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material was not readily biodegradable under the conditions of the modified Sturm test.

Executive summary:

The ready biodegradability of the test material was assessed in a modified Sturm test conducted in accordance with the standardised guidelines OECD 301B, EU Method C.4-C, ISO 9439 and ISO 10634 under GLP conditions.

The test material was tested in duplicate at a target concentration of 16 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of the test material was calculated to be 2.78 mg CO2/mg.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 positive control (sodium acetate) and 1 toxicity control (test material plus sodium acetate).

Since the test material 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 material. 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 material and test medium. Furthermore, the test medium was daily swirled around, since the test material tended to float on the water surface. Test duration was 28 days (last CO2-measurement on day 29).

The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 9 % biodegradation of the test material (based on ThCO2) for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation within a 10-day window) was not met. In the toxicity control, the test material was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

The test material was not readily biodegradable under the conditions of the modified Sturm test.

Description of key information

The test material was not readily biodegradable under the conditions of the modified Sturm test.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test material was assessed in a modified Sturm test conducted in accordance with the standardised guidelines OECD 301B, EU Method C.4-C, ISO 9439 and ISO 10634 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in duplicate at a target concentration of 16 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of the test material was calculated to be 2.78 mg CO2/mg.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 positive control (sodium acetate) and 1 toxicity control (test material plus sodium acetate).

Since the test material 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 material. 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 material and test medium. Furthermore, the test medium was daily swirled around, since the test material tended to float on the water surface. Test duration was 28 days (last CO2-measurement on day 29).

 The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 9 % biodegradation of the test material (based on ThCO2) for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation within a 10-day window) was not met. In the toxicity control, the test material was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

The test material was not readily biodegradable under the conditions of the modified Sturm test.