Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-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:

20 February 2018 to 07 June 2018

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 1063

Version / remarks:

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 (adaptation not specified)

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.
- The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm). After treatment, the concentration of suspended solids (SS) was determined to be 3 g/L in the concentrated sludge as used for the test. 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 8 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

(mg TOC/L)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Milli- RO water: Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
Stock solutions of mineral components:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O and 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.
Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
- Additional substrate: No. On the day of testing weighed amounts of the test material were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test material bottle A: 30.04 mg; test material bottle B: 30.01 mg and toxicity control bottle: 29.88 mg). To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test material and the test organisms. Any residual volumes were discarded.
- Test temperature: 22-23 °C
- pH: 7.6-7.8
- pH adjusted: yes, adjusted using 1 M HCl
- Aeration of dilution water: During the test period, the test media were aerated and stirred continuously.
- Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
- 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 passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- 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.
- Number of culture flasks/concentration: 2
- 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. 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.
- 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 standardised HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

SAMPLING
- Measurements: 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 material. Titrations for the procedure and toxicity control were made over a period of at least 14 days.
- 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 respective test suspensions was measured and 1 mL of concentrated HCl (37 %, Merck) 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 15 (procedure and toxicity control) and on day 29 (remaining vessels).
- Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.
- pH: At the start of the test (day 0) and on the penultimate day (day 14 for the procedure and toxicity control and day 28 for the inoculum blanks and test material), before addition of concentrated HCl.
- Temperature of medium: Continuously in a vessel with Milli- RO water in the same room.

CONTROL AND BLANK SYSTEM
- Test suspension: containing test material and inoculum (2 bottles)
- Inoculum blank: containing only inoculum (2 bottles)
- Procedure control: containing reference material and inoculum (1 bottle)
- Toxicity control: containing test material, reference material and inoculum (1 bottle)

STATISTICAL METHODS:
- ThCO2, expressed as mg CO2/mg test material, was calculated from the results of carbon analysis:
ThCO2 = (Fraction organic carbon × 44) / 12
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 M HCl titrated is converted into mg of CO2 produced:
mg CO2 = [(0.05 x Δ mL HCl titrated)/2] x44 = 1.1 x Δ mL HCl titrated
- Relative biodegradation values were calculated from the cumulative CO2 production relative to the ThCO2. A figure of more than 10 % biodegradation was considered biologically relevant. The relative biodegradation values were plotted versus time together with the relative biodegradation of the procedure control. Assessment of ready biodegradability was made based on the average biodegradation in test material bottle A and B. If not clear from the experimental data the number of days was calculated from the attainment of 10 % biodegradation until 60 % biodegradation. If this period was ≤ 10 days (10-day window) the test material was designated as readily biodegradable.
- Toxicity control: if less than 25 % biodegradation (based on the combined ThCO2 of the test material and reference material) 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:

18

Sampling time:

28 d

Remarks on result:

other: Mean of Bottles A and B

Details on results:

Theoretical CO2 Production
- The ThCO2 of the test material was calculated to be 2.89 mg CO2/mg.
- The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation
- The relative biodegradation values calculated from the measurements performed during the test period revealed 11 % and 24 % biodegradation of the test material (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation window) was not met.
- In the toxicity control, more than 25 % biodegradation occurred within 14 days (40 %, based on ThCO2). Therefore, the test material was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Monitoring of Temperature and pH
- The temperature recorded in a vessel with water in the same room varied between 22 and 23 °C.
- pH was from 7.6-7.8

Results with reference substance:

- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Table 1: Biodegradation of the Test Material in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	Δ A-B *
2	0	0	0	0
5	1	5	3	4
8	3	11	7	8
13	6	14	10	8
15	6	17	12	11
19	7	19	13	12
23	8	19	14	11
29**	10	21	16	11
29**	11	23	17	12
29**	11	24	18	13

*Absolute difference in biodegradation between bottles A and B

** Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material was not readily biodegradable.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301B, under GLP conditions.

The test material was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test material was determined to be 78.77 %. Based on the TOC content the ThCO2 of the test material was calculated to be 2.89 mg CO2/mg. The test material was tested in duplicate at a target concentration of 15 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 procedure 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-litres test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test material and test organisms. Test duration was 28 days for the inoculum blank and test material (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 the test period revealed 11 % and 24 % biodegradation of the test material (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation) 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.

Under the conditions of this study, the test material was not readily biodegradable.

Description of key information

Under the conditions of the study, the test material was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301B, 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 not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test material was determined to be 78.77 %. Based on the TOC content the ThCO2 of the test material was calculated to be 2.89 mg CO2/mg. The test material was tested in duplicate at a target concentration of 15 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 procedure 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-litres test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test material and test organisms. Test duration was 28 days for the inoculum blank and test material (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 the test period revealed 11 % and 24 % biodegradation of the test material (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60 % biodegradation) 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.

Under the conditions of the study, the test material was not readily biodegradable.