Oxacycloheptadecan-2-one

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:

12 December 2016 - 09 March 2017 (Two experiments were performed. However, the first experiment was disregarded since the difference between replicate bottle A and B was >20%. Only the second experiment is reported).

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:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1999

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 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

Specific details on test material used for the study:

- Appearance: Colourless to very pale yellowish solid
- Test item storage: In refrigerator (2-8°C)
- Solubility in water: Insoluble
- Stability in water: Stable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Storage conditions/Storage length: The freshly obtained sludge was kept under continuous aeration for approximately 24 hours, until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (55 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- 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: 4.8 g suspended solids/L in the concentrated sludge
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

16 mg/L

Based on:

test mat.

Remarks:

(nominal)

Initial conc.:

44.3 mg/L

Based on:

ThCO2

Remarks:

(based on the nominal initial concentration of 16 mg/L and a ThCO2 of 2.77 mg CO2 per mg test substance)

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: according to OECD TG 301A/B
- Pre-incubation of 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.
- Preparation of test concentrations: Since Oxacycloheptadecan-2-one 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 (test item bottle A: 31.8 mg; test item bottle B: 32.1 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 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 the test organisms.
- Preparation of reference control: A solution of sodium acetate was prepared by dissolving 800.3 mg in Milli-RO water and making this up to a total volume of 200 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).
- Test temperature: The temperature, continuously recorded in a vessel with water in the same room, varied between 22.0 and 23.3 °C.
- pH: At the start of the test te pH was 7.6 in all test media except the toxicity control in which it was 7.5. At day 28 (before addition of HCl for titration) the pH varied from 7.5 to 7.8.
- pH adjusted: yes. At the start of the test pH was adjusted using 1 M HCl in case it was >7.6.
- Continuous darkness: yes (test media excluded from light)

TEST SYSTEM
- Culturing apparatus: 2 litre glass brown coloured bottles.
- Number of culture flasks/concentration: 2 test suspensions (containing test item and inoculum); 2 inoculum blanks (containing only inoculum); 1 positive control (containing reference item and inoculum); 1 toxicity control (containing test item, reference item and inoculum).
- Preparation of culture flasks: 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.
- Aeration: Continuous with synthetic air (a mixture of ca. 20% oxygen and ca. 80% nitrogen with CO2 < 1 ppm).
- Details of trap for CO2: Synthetic air 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).

SAMPLING
- Sampling frequency: Titrations were made on days 2, 5, 7, 9, 12, 14, 19, 23, 27 and 29 for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made on days 2, 5, 7, 9, 12 and 14.
- Sampling method: 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, Merck) was used as pH-indicator. On day 28, the pH of all 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.
- Other: 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, Merck) was used as pH-indicator.

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

Reference substance:

acetic acid, sodium salt

Remarks:

(40 mg/L based on test mat., 1.07 mg/L based on ThCO2)

Test performance:

In the toxicity control, more than 25% biodegradation occurred within 14 days (49%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

64

Sampling time:

28 d

Details on results:

The relative biodegradation values calculated from the measurements performed during the test period revealed 63% and 65% biodegradation in the test replicates (bottles A and B, respectively). However, biodegradation of the test substance of at least 60% was not reached within a 10-day window. Thus, the criterion for ready biodegradability was not met.

Results with reference substance:

The reference item sodium acetate was biodegraded for 67% within 14 days and showed a normal biodegradation curve.

Table: CO2 produced in the blank

Day	HCl (0.05 N) titrated (mL)		Produced CO2		Cumulative CO2 (mg)
	Ba(OH)2 *	Blank ***	mL HCl	mg
2	48.58	45.88	2.70	3.0	3.0
5	48.44	44.51	3.97	4.3	7.3
7	48.73	44.41	4.33	4.8	12.0
9	48.89	44.57	4.33	4.8	16.8
12	48.51	44.72	3.79	4.2	21.0
14	48.63	44.48	4.15	4.6	25.5
19	48.61	43.88	4.73	5.2	30.7
23	49.97	45.09	4.89	5.4	36.1
27	50.59	44.20	6.39	7.0	43.1
29 *	50.00	44.52	5.49	6.0	49.2
29*	49.80	46.55	3.25	3.6	52.7
29*	49.50	48.60	0.91	1.0	53.7

*    CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl

**  “Strength” of untreated 0.0125 M Ba(OH)2 solution

*** Mean value of two replicates

 

Table: CO2 production and percentage biodegradation of the test item (Bottle A)

Day	HCl (0.05 N) titrated (mL)		Produced CO2		Cumulative CO2 (mg)	Biodegradation   (%) **
	Blank	Bottle A	mL HCl	mg
2	45.88	46.43	0.00	0.0	0.0	0
5	44.51	40.81	3.69	4.1	4.1	5
7	44.41	41.32	3.09	3.4	7.5	8
9	44.57	39.96	4.61	5.1	12.5	14
12	44.72	34.31	10.41	11.5	24.0	27
14	44.48	35.41	9.07	10.0	33.9	39
19	43.88	33.23	10.65	11.7	45.7	52
23	45.09	39.95	5.14	5.6	51.3	58
27	44.20	42.08	2.12	2.3	53.6	61
29*	44.52	42.98	1.54	1.7	55.3	63
29*	46.55	47.18	0.00	0.0	55.3	63
29*	48.60	48.60	0.00	0.0	55.3	63

*  Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 88.1 mg CO2/2L

** CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table: CO2 production and percentage biodegradation of the test item (Bottle B)

Day	HCl (0.05 N) titrated (mL)		Produced CO2		Cumulative CO2 (mg)	Biodegradation   (%) **
	Blank	Bottle B	mL HCl	mg
2	45.88	46.68	0.00	0.0	0.0	0
5	44.51	41.28	3.22	3.5	3.5	4
7	44.41	40.22	4.19	4.6	8.2	9
9	44.57	38.00	6.57	7.2	15.4	17
12	44.72	36.15	8.57	9.4	24.8	28
14	44.48	36.74	7.74	8.5	33.3	37
19	43.88	33.46	40.42	11.5	44.8	50
23	45.09	40.56	4.53	5.0	49.7	56
27	44.20	40.96	3.24	3.6	53.3	60
29*	44.52	40.96	3.56	3.9	57.2	64
29*	46.55	46.21	0.00	0.0	57.2	64
29*	48.60	48.17	0.42	0.5	57.7	65

*  Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 88.9 mg CO2/2L

** CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table: CO2 produced and percentage biodegradation in the positive control (PC)

Day	HCl (0.05 N) titrated (mL)		Produced CO2		Cumulative CO2 (mg)	Biodegradation   (%) *
	Blank	PC	mL HCl	mg
2	45.88	45.93	0.00	0.0	0.0	0
5	44.51	24.73	19.78	21.8	21.8	25
7	44.41	35.68	8.73	9.6	31.4	37
9	44.57	34.99	9.58	10.5	41.9	49
12	44.72	35.17	9.55	10.5	52.4	61
14	44.48	40.05	4.43	4.9	57.3	67

* Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 mg CO2/2L

 

Table: CO2 produced and percentage biodegradation in the toxicity control (TC)

Day	HCl (0.05 N) titrated (mL)		Produced CO2		Cumulative CO2 (mg)	Biodegradation   (%) *
	Blank	TC	mL HCl	mg
2	45.88	41.95	3.93	4.3	4.3	2
5	44.51	19.22	25.29	27.8	32.1	18
7	44.41	30.67	13.74	15.1	47.2	27
9	44.57	28.78	15.79	17.4	64.6	37
12	44.72	31.56	13.16	14.5	79.1	45
14	44.48	38.89	5.59	6.1	85.2	49

* Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive

control: 174.5 mg CO2/2L (ThCO2 test item: 88.9 mg CO2/2L + ThCO2 sodium acetate: 85.6 mg CO2/2L)

 

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks'

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The substance biodegraded >60% within 28 days but the 10-day window criterion was not met. The substance is qualified as 'readily biodegradable, but failing 10-day window.

Executive summary:

The biodegradation potential of the substance was examined in a study according to OECD TG 301B (Modified Sturm test) and in compliance with GLP criteria. About 16 mg/L test substance (44.3 mg/L based on ThCO2) was inoculated for 28 days under aerobic conditions and in the dark with activated sludge from a municipal sewage treatment plant receiving predominantly domestic sewage. After 2, 5, 7, 9, 12, 14, 19, 23, 27 and 29 days inoculation the produced CO2 was measured by precipitation with barium hydroxide and titration of the remaining barium hydroxide with HCl. Two replicate bottles were used for the test item and inoculum blank. A reference control and toxicity control were run in parallel. After 28 days inoculation, the substance was biodegraded for 64% (mean value). However, biodegradation of the test substance of at least 60% was not reached within a 10-day window and thus the criterion for ready biodegradability was not met. Based on these findings the substance is qualified as readily biodegradable, but failing 10 -day window.

Description of key information

The biodegradation potential of the substance was examined in a study according to OECD TG 301B (Modified Sturm test) and in compliance with GLP criteria. About 16 mg/L test substance (44.3 mg/L based on ThCO2) was inoculated for 28 days under aerobic conditions and in the dark with activated sludge from a municipal sewage treatment plant receiving predominantly domestic sewage. After 2, 5, 7, 9, 12, 14, 19, 23, 27 and 29 days inoculation the produced CO2 was measured by precipitation with barium hydroxide and titration of the remaining barium hydroxide with HCl. Two replicate bottles were used for the test item and inoculum blank. A reference control and toxicity control were run in parallel. After 28 days inoculation, the substance was biodegraded for 64% (mean value). However, biodegradation of the test substance of at least 60% was not reached within a 10-day window and thus the criterion for ready biodegradability was not met. Based on these findings the substance is qualified as readily biodegradable, but failing 10 -day window.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

A BIOWIN calculation was performed to support 'rapid degradation' with respect to environmental classification.

The substance is assessed to be 'readily biodegradable' based on BIOWIN modules 1,2,3,5 and 6.