3,20-bis(ethylenedioxy)pregna-5,7-diene

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-23 to 2014-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In accordance with OECD and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

solid: crystalline

Details on test material:

Name of test material (as cited in study report): Proketal
- Molecular formula C25H3504
- Substance type: Crystalline
- Physical state: Solid
- Stability under test conditions: Unstable after repeated exposure to air; hydrolysis; stable in the light
- Storage condition of test material: Keep in a well-closed container in dry and well-venti lated place at room temperature (15-25 °C)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

Sludge: city sewage plant (JiangQiao Wastewater Treatment Plant, JinShan Wastewater Marine Disposal Treatment Plant, LangXia Wastewater Treatment Plant), Industry sewage plant (Sino French water)
The sludge, water and soil were sampling from 10 sites on Aug 29th 2013

The inoculum consisted of sludge, water and soil which was sampled from multiple sites to cover for different environmental conditions to which the test substance may be exposed is based on the requirments of "OECD, Guidelines for the Testing of Chemicals, 1981, 302C Inherent Biodegradability: Modified MITI Test (II)". Characteristics which make the above inoculum suitable for 302C are their higher activity .

All samples (sludge, river and sea water) were mixed by stirring in a single container and the mixture is transferred into a culture tank after removing the floating particles and debris. The culturing is aerated and carried out at 25±2°C and adjusted to pH7.0± 1.0. Approximately one third of the whole volume of the supernatant was removed and an equal volume of synthetic sewage was added to the reaining portion of the supernatant everyday.

BOD determination began on Oct 24th 2013. A fresh sample of activated sludge was collected from the culture tank, cleaned and washed 3 times with mineral medium. The sludge was seperated by centrifuging for 20 minutes at ~4000rpm. A small amount of the washed sludge was weighed and dried to calculate the water content (105°C). The dry weight was 5.18%.
Wet sludge was suspended in the mineral culture medium to obtain a concentration of 2g dry matter/L. The sludge suspension was used as the inoculum after 30min stirring at 500rpm. The concentration of the sludge in the fibal test solution was 100mg dry matter/L

The sludge cultured for 2 months was used as the inoculum in Modified MITI Test II.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test substance was directly added to the test solution and the nominal concentration of test substance was about 30 mg/L. No emulsifiers or solvents were
used.

To prepare the stock solution of reference substance, 0.50050 g of sodium benzoate was dissolved in 250 ml mineral medium and the concentration of stock solution was 2 g/L. The final concentration of reference substance in the test system was 100 mg/L.

The following test system were used:
blank control group (18), (sludge only)
procedure control group (PC), (reference substance)
test substance group (TS),
toxicity control group (TC) (test and reference substance)
abiotic control group (AC) (test substance and water)

Brown bottles with the volume of 500 ml were used.

BOD determination
WTW Oxitop Operational procedure as follows:
1) Estimate the measuring range of the test substance to be analyzed. Before filling
the BOD bottles, add all the additional solution as above.
2) Insert a magnetic stirrer bar into the bottle.
3) Place 2 sodium hydroxide pellets in the rubber sleeve.
4) Insert the rubber sleeve onto the bottle.
5) Screw on the OxiTop® measuring head tightly.
6) Start the measurement on the OxiTop® head.
7) Place the overflow measuring flask in the incubator for 28 days at 25+2°C.
Stirring, temperature and data, are periodically inspected. Final results are recorded after 28 days.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

During the 28-d test, the temperature was in the range of 23.6-24.6°C. The air-tightness of the test systems were well and magnetic stirrers were running well.
The pH of test solutions were within the range of 6.515-7.253.
The degradation percentage of toxicity control on day 14 was 48.68%, which was higher than the toxicity inhibition level (>25%). It is concluded that the test substance has no inhibitory effects on inoculum at the concentration of 30 mg/L.

All the results indicated that no mineralization occurred during the 28 days (the test substance could not be totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts etc.).

No oxygen consumption could be observed in abiotic control in the day 28, the calculation results showed that there was no aerobic abiotic degradation '

Method validation

According to the cumulative oxygen consumption, the mean degradation percentage of the reference substance were 80.06% and 83.62% after 7 and 14 days, respectively. Quality control requirements of the test are that the degradation percentage of the reference substance exceeds 40% and 65% after 7 days and 14 days, respectively. Hence, the test is considered to be valid.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

2.09

Sampling time:

28 d

Details on results:

The results of biochemical oxygen demand (BOD) indicated that the cumulative oxygen consumption level of three replicates of test substance group were 88.80 mg/L , 80.30 mg/Land 88.80 mg/L respectively. They were very similar as the mean oxygen consumption of blank controls (85.95 mg/L). The calculated percentage biodegradation based on the BOD determination was 0.02% at the 28d which showed that this test substance is not biodegradable.
The mean percentage biodegradation of test substance in the 28th day was 2.09% based on the calculated results of UPLC analysis. The test substance was not proved to be inherent degradable under the present test conditions.

BOD5 / COD results

BOD5 / COD

Parameter:

BOD5

Value:

0.02 other: %

Results with reference substance:

the mean cumulative oxygen consumption of blank controls 85.95 mg/L

Any other information on results incl. tables

The mean measured concentration of test substance group (mean value) and measured concentration of abiotic control group were 24.5 mg/L and 24.0 mg/L respectively at zero-time, 23.4 mg/L and 23.9 mg/L respectively on day 28. The mean residual rate of test substance group was 95.51 % on day 28, and the residual rate of abiotic control was 99.58%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The percentage biodegradation were calculated to 0.02% and 2.09% respectively based on the results of BOD and UPLC analysis. The test substance was found to be non-inherent biodegradable under the present test conditions.