Pregna-1,4,6-triene-3,20-dione, 17-(acetyloxy)-

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Mar 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

17-Acetoxy-1,4,6-pregnatriene-3,20-dione

EC Number:

607-988-8

Cas Number:

2668-75-9

Molecular formula:

C23 H28 O4

IUPAC Name:

17-Acetoxy-1,4,6-pregnatriene-3,20-dione

Study design

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 activated sewage sludge for the inoculum was collected on the day before the start of the experiment from a well-operated municipal sewage treatment plant ("Kläranlage Berlin-Ruhleben"), predominantly dealing with domestic sewage. Upon arrival at the laboratory, the activated sludge was stirred and aerated tor approximately 3 hours. Thereafter, the sludge was homogenized in a blender. Then it was allowed to settle tor approximately 1 hour. 24 mL (90 mg) suspended solids were taken from the supernatant for each test vessel.

Duration of test (contact time):

36 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

DOC

Details on study design:

TEST CONDITIONS
The test solutions were incubated at 20 to 24°C for 36 days (except on day 29 when the room temperature rose to 28 °C for a period of approximately 16 hours after wh ich the room temperature decreased to 17 -18 °C and increased to a normal temperature of 20-21 °C on day 32 due to technical problems with the air-conditioning system). Furthermore, the test solutions were supplied with filtered CO2-free aeration (2.0 to 7.5 L air per hour for each test vessel) for 36 days. On day 35 the pH of the test solutions, including reference and blank control, ranged from 7.6 to 8.0. After the measurements of pH on day 35,1 mL of concentrated HCL was added to each test vessel in order to convert all dissolved inorganic carbon into CO2.


TEST SYSTEM
For the determination of the CO2 produced, thme CO2 absorber bottles, filled with 100 ml 0.025 N Ba(OH)2 each, were connected in series to the exit air-pipe of each test bottle. The amount of CO2 produced was determined by titration of the remaining Ba(OH)2 with 0.05 N standardised HCI. On days 3, 4, 7, 9, 11, 14, 18, 23, 28, 30, 32, 35 and 36 the CO2 absorber bottle nearest to the test bottles was removed for the titration. The remaining two absorbers were each moved one place closer to the test vessel and a new absorber bottle filled with fresh Ba(OH)2 was placed at the far end of the series. In cases where BaCO3 was also precipitated in the second absorber bottle of any solution, titrations of two Ba(OH)2 bottles were performed. Subsequently, two fresh absorbers were added.

24 mL of the inoculum from the activated sludge were added to the nutrient solutions in each of the test vessels. These mixtures were aerated wiith CO2-free air for about 24 hours to purge the system of carbon dioxide before test and reference compounds were added. CO2-free air was obtained by bubbling filtered air through activated charcoal and soda lime pellets. A stock solution of the reference substance was prepared by dissolving 1.0 g sodium acetate in 1 L demineralized water. 102 mL of this stock solution were added to the reference test bottle and into the bottle for the toxicity controI. The test material was dispersed weil in vessels containing approximately 1 mL water and added directly at amounts of 40.0 mg ZK 5560 to each of three test bottles and one bottle for the toxicity control. Finally, the test vessels were rinsed with 2-3 mL demineralized water. Finally, each test vessel was filled up with deminelfalized water to a volume of 3000 mL. Thus, the concentrations (based of carbon) of the reference and the test compounds were as folIows:
ZK 5560: 10 mg/L
reference substance (sodium acetate): 10 mg/L
toxicity control (ZK 5560 + sodium acetate): 10 mg/L + 10 mg/L
Three test bottles contained only nutrient reagents and inoculum and served as blank (control) vessel.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

71

Sampling time:

34 d

Remarks on result:

other: failed 10 day window

Details on results:

After a relatively long lag-phase (until day 18) the test compound was degraded rapidly to 71% on day 36 (34 days of incubation).

BOD5 / COD results

Results with reference substance:

The reference compound sodium acetate produced 99 mg CO2 which was equivalent to 90% of the theoretical CO2 production.

Any other information on results incl. tables

Table 1: Biological degradation (cumulative) in percent (corrected for blank CO2 production) of Delta-1,6-Hydroxyprogesteroncetat

Test	Nominal	Day of sampling
compound	concentration of carbon	3	4	7	9	11	14	18	23	28	30	32	35
ZK 5560	10 mg/l	0	0	0	1	1	1	2	18	45	59	66	69
Sodium acetate (reference)	10 mg/l	15	33	58	68	73	80	84	87	89	88	88	89
ZK 5560 +	10 mg/l +	7	15	27	43	54	67	76	82	84	85	86	86
sodium acetate	10 mg/l
(toxicity control)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The test compound Delta-1,6-Hydroxyprogesteronacetat is not readily biodegradable according to the OECD criteria but considered being biodegradable based on the high amount degraded at the end of the study.

Executive summary:

The test substance Delta-1,6-Hydroxyprogesteronacetat was incubated in an aqueous solution including nutrients with microorganisms from a municipal sewage treatment plant for 34 days (start of treatment = day 1). The experiment was extended to 34 days because the plateau was not reached by day 28. The nutrient solutions were made up of phosphates, ammonium sulphate, magnesium sulphate, iron chloride, ammonium chloride and calcium chloride and added to the test solution. The test substance was incubated in a c:oncentration of 10 mg carbon/L in triplicate. Additionally, a reference substance (sodium acetate) was tested according to the same procedure, in order to verify the viability and activity of the degrading microorganisms. Furthermore, a blank control was tested in triplicate without any test or reference substance. One further set was incubated with sodium acetate as 10 mg carbon/L (reference substance) plus test substance as 10 mg carbon/L representing a toxicity control. The biological degradation of the test and reference substances was evaluated by measurement of the carbon dioxide (C02) produced during the test period. CO2 production was determined on days 3, 4, 7, 9, 11, 14, 18, 23, 28, 30, 32, 35 and 36 and calculated as the percentage of total CO2 that the test material could theoretically have produced, based on carbon content. The blank CO2 production was subtracted for correction. After a relatively long lag-phase (until day 18) the test compound Delta-1,6-Hydroxyprogesteronacetat was degraded rapidly to 71% on day 36 (~ 34 days of incubation).

The reference compound sodium acetate was degraded to 73% on day 11 (~ 10 days of incubation) and up to 90% on day 36.

In the toxicity control, the reference compound (sodium acetate) plus the test compound, was degraded to 87% on day 36 (~ 34. days of incubation). The degradation of the toxicity control seemed to start on days 9 to 11, earlier than in the individual set, indicated by the higher degradation in the toxicity control compared to the individual sets.

The test compound Delta-1,6-Hydroxyprogesteronacetat is not readily biodegradable based on the OECD criteria since the 60% trigger was not reached after 28 d of incubation. However, the substance is considered biodegradable to a high extent since 71% were degraded after approx. 34 d of incubation.