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

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-08-28 to 2014-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Per OECD method 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 305 (Bioconcentration: Flow-through Fish Test)

GLP compliance:

yes

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

Sampling and analysis

Details on sampling:

- Sampling intervals/frequency for test organisms: Test fish sampling were determined on 3d and 13d of the uptake phase and 1d, 3d, 5d, 7d, 14d, 21 d and 28d of the depuration phase.
- Sampling intervals/frequency for test medium samples: test diets and control diets before and after each renewal and at the end of the uptake phase.
- Sample storage conditions before analysis: Fish samples were placed in culture dishes and stored at refrigerator(4 'C, 15min) for anaesthesia. They were weighed and placed in a -80°C freezer for analyzing.

The concentrations of the test substance in the test water were determined before the bioaccumulation test, on 0d, 7d and 14d of the uptake phase and 7d, 14d, 21d and 28d of the depuration phase.

Length, body weight and lipid determination

The length and body weight of fish were measured in random on twenty fish from the the stock population at the start of the uptake phase. And twenty fish from the control and each treatment were measured separately at the end of the depuration phase.

The lipid content of the fish was determined on six fish from the stock population at the start of the uptake phase. And six fish from the control and each treatment were determined at the end of the uptake phase and the depuration phase

Test solutions

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

The test substance was dissolved in acetone to prepare the stock solutions which were used to prepare the test diets with dosages of 50.0 mg/kg and 100 mg/kg. The test diets were then dried overnight. Based on stability testing the stock solutions and the test diets were renewed every 9 days during uptake phase.

Test organisms

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

Before being used in the test, all fish were acclimatized in the following test conditions for more than 14 days:

The test was performed with Gobiocypris rarus which were bred in Bioassay and Safety Assessment Lab, Shanghai Academy of Public Measurement. Before being used in the test, all fish were acclimatized in the following test conditions for more than 14 days:

At the beginning of the test, length was 2.3±0.5cm(Mean ± SD, the same as below), weight was 0.24±0.049. At the end of the test, the length and weight of fish for the control group were 2.6±0.2cm and 0.27±0.059, respectively. The length and weight of fish for the 50mg/kg treatment group were 2.6±0.2cm and 0.28±0.059, respectively.The length and weight of fish for the 100 mg/kg treatment group were
2.6±0.2cm and 0.29±0.079, respectively.

Lipid content of the fish body weight in the control group at the beginning of the test was 10.6%.

Feeding: fish were fed twice daily with Brine Shrimp Aquarium Flake Food, O.S.I®, which crude protein content and crude fat content are greater than 48% and 7%, respectively.

Study design

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

28 d

Test conditions

Hardness:

Water: the test water used was tap water dechlorinated by activated carbon and sterilized by UV lamp;

Test temperature:

Temperature: 21-25'C and constant within a range of 2'C.
conditions of exposure: 21.8 to 23.8 C

pH:

pH: 6.0-8.5;

Dissolved oxygen:

Oxygen concentration: >80% of air saturation value(ASV)
conditions of exposure: Oxygen concentration: 62.1 %-91.3% ASV.

TOC:

Total organic carbon: not detected; 1.88 mg/L

Salinity:

145mg CaCO3/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tanks
- Material, size, headspace, fill volume: 18 L
- Aeration: yes continuous
- Type of flow-through: peristaltic pump system
- Renewal rate of test solution (frequency/flow rate): st 52.08 mL/min; Approximately 75 litres of the test water flowed through each test chamber per 24 hours.
- No. of organisms per vessel: 50
- No. of vessels per concentration (replicates): 2
- No. of vessels per control / vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS

The water was aerated continuously over 24h and kept temperature with 25 ± 1 'C. Water quality parameters were as follows:

Total hardness: 145mg CaCO3/L; Residual chlorine: not detected; .

- Source/preparation of dilution water: Tap water, dechlorinated by activated carbon and sterilized by UV lamp, was used.
- Particulate matter: 4mg/L
- NITRITE (N02--N)): 0.006mg/L;
- NITRATE(N03--N): 1.2mg/L
- Chlorine: not detected
- Alkalinity: 145mg CaCO3/L

OTHER TEST CONDITIONS:

Photoperiod: Light/Dark=12hours/12 hours;

Nominal and measured concentrations:

Loading: at the beginning of the test, it was 0.2g fish/litre per day; the nominal concentrations in the test diets of the treatment groups were 50.0mg/kg and 100 mg/kg within the range of 1-100 mg/kg. A control group of test water was also used. Each group had two replicates.

Results and discussion

Lipid content

Lipid content 1

Lipid content:

10.6 %

Time point:

start of exposure

Lipid content 2

Lipid content:

11.3 %

Time point:

end of exposure

Remarks on result:

other: control

Lipid content 3

Lipid content:

10.8 %

Time point:

end of exposure

Remarks on result:

other: 50.0 mg/kg

Lipid content 4

Lipid content:

9.79 %

Time point:

end of exposure

Remarks on result:

other: 100 mg/kg

Lipid content 5

Lipid content:

9.58 %

Time point:

other: end depuration phase

Remarks on result:

other: control

Lipid content 6

Lipid content:

11.2 %

Time point:

other: end depuration phase

Remarks on result:

other: 50.0 mg/kg

Lipid content 7

Lipid content:

11.5 %

Time point:

other: end depuration phase

Remarks on result:

other: 100 mg/kg

Bioaccumulation factor

Bioaccumulation factor 1

Type:

BMF

Value:

0.049 dimensionless

Basis:

normalised lipid fraction

Calculation basis:

kinetic

Remarks on result:

other: Conc.in environment / dose:50.0 mg/kg

Bioaccumulation factor 2

Type:

BMF

Value:

0.051 dimensionless

Basis:

normalised lipid fraction

Calculation basis:

kinetic

Remarks on result:

other: Conc.in environment / dose:100 mg/kg

Details on kinetic parameters:

At a dynamic-state, the overall depuration rate constants for 50.0mg/kg groups(two replicates) were 0.453 and0.450, and the assimilation efficiency were 0.983 and 0.565, respectively. The overall depuration rate constants for 100mg/kg groups (two replicates) were 0.487 and 0.466, and the assimilation efficiency were 0.759 and 0.754, respectively. Biomagnification factors at a dynamic-state(BMFk) of the 50.0mg/kg group(the geometric mean measured concentration in the test diets was 49.5mg/kg) and 100mg/kg group( the geometric mean measured concentration in the test diets was 90.9mg/kg) to Gobiocypris rarus were 0.0257 and 0.0239, and the lipid-corrected kinetic dietary biomagnification factor(BMFL) were 0.0492 and 0.0505 for the spiked 50.0mg/kg and 100mg/kg samples, respectively.

Details on results:

The test fish were exposed to the 50.0mg/kg and 100mg/kg treatments respectively for 14 days(i.e. uptake phase), and the measured concentrations of test substance in test fish were gradually increased. At the end of the uptake phase, the measured concentrations for 50.0mg/kg treatments were 1.61mg/kg and 0.930mg/kg, the measured concentrations for 100mg/kg treatments were 2.13mg/kg and 2.21mg/kg. During the depuration phase, the measured concentrations in test fish were gradually reduced, at the Day 21 of depuration phase, the measured concentrations of the test substance in test fish were lower than the LOD4(LOD4, LOD4=55.5ng).

The mean weight of the test fish was 0.24g at the beginning of the uptake phase. At the end of the depuration phase, the mean weight of the test fish of the control group, 50mg/kg and 100mg/kg group were 0.27g, 0.28g and 0.29g, respectively.

The lipid content of the test fish was 10.6% at the start of the uptake phase. At the end of the uptake phase, the lipid content of the test fish of the control group, 50mg/kg and 100mg/kg group were 11.3%, 10.8% and 9. 79%, respectively. At the end of the depuration phase, the lipid content of the test fish of the control group, 50mg/kg and 100mg/kg group were 9.58%, 11.2% and 11.5%, respectively. At the end of the test, the coefficient of variations on the lipid content were 7.15%, 3.64% and 5.45% in each group separately, which compared with that at the beginning. They were less than 10%. The mean lipid fraction in the food measured at exposure start and end were 20.5% and 20.8%, and the mean lipid fraction in the fish of the treatment groups measured at end of exposure were 10.8% and 9.79%, respectively. The lipid-correction factor were 0.522 and 0.473 for the spiked 50.0mg/kg and 100mg/kg samples respectively.

Any other information on results incl. tables

Analysis of the test substance in test water: It was found that the average recoveries were 104% and 91.6%  of the spiked 222ng/ml  and 3330ng/ml  samples with RSD (relative standard deviation) values of 1.25% and 3.56%, respectively. In bioaccumulation test,the test substance in the test water of all the groups were not detected. 

Analysis of the test substance in test fish: average recoveries were 93.4 % and 96.3% of the spiked 222 ng and 1670 mg samples with RSD values of

4.82% and 7.93%,  respectively. During the uptake the measured concentration varied by less than 20% and therefore the geometric mean measured concentrations were used.

Analysis of the test substance in test diets: average recoveries were 90.1 % and 87.9% of the spiked 55.5 mg/kg and 111 mg/kg samples with RSD values of 4.91% and 8.65%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the tested conditions, biomagnification factors at a dynamic-state(BMFk) of 50.0mg/kg group (the geometric mean measured concentration in the test diets was 49.5mg/kg) and 100 mg/kg group (the geometric mean measured concentration in the test diets was 90.9mg/kg) to Gobiocypris rarus were 0.0257 and 0.0239, and the lipid-corrected kinetic dietary biomagnification factor (BMFL) were 0.0492 and 0.0505 for the spiked 50.0mg/kg and 100mg/kg samples, respectively.