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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 February 2013 to 27 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In accordance with GLP and OECD methods. Substance is well characterised.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

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

Details on test material:

Proketal; pregna-5,7-diene-3,20-dione, cyclic 3,20-bis(1,2-ethanediyl acetal)
-Molecular weight 400.5
-Purity/composition correction No factor required
-Hygroscopic Not indicated
-Volatile No
-Stability in water Not indicated
-Solubility in water <0.01 mg/L

- Substance type:
- Physical state: Slightly yellow crystalline powder
Stability under test conditions: Stable
- Storage condition of test material: At room temperature (<25C) in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for analytical confirmation of actual exposure concentrations were taken at the start and after 48-hours of exposure.

Volume 2 mL from the approximate center of the test vessels
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, singular reserve samples of 2 mL were taken for possible analysis. If not used,these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Details on test solutions:

Stock and spiking solutions
Stock solutions of the test substance were prepared in acetonitrile at concentrations of 584 and 976 mg/l. In order to dissolve the test substance the solutions were ultrasonicated for 10 minutes.

Spiking solutions were prepared in tetrahydrofuran. In order to dissolve the test substance the solutions were ultrasonicated for 5 minutes.

Calibration solutions
Calibration solutions in the concentration range of 0.04 – 60 mg/l were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.

Procedural recovery samples
2 ml blank medium was spiked with the test substance at a target concentration of 0.1 or
100 mg/l. The accuracy samples were treated similarly as the test samples (see paragraph 4.2 ‘Samples’).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Crustacea, Cladocera
- Source: Straus, 1820
Daphnids of at least the third generation and obtained by acyclical parthenogenesis under specified breeding conditions were used.
The batch was considered valid if the daphnids originated from a healthy stock; being the 2nd to 5th brood of the parental batch which showed no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood was observed. For the test young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old were selected.


ACCLIMATION
A new batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. The maximum age of the culture was 4 weeks and half of the medium (M7) was renewed after the first seven days of cultivation. After this renewal, half of the medium was renewed twice a week. The temperature of the medium was 18-22°C. Daphnids were fed daily with a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Methods: A static combined limit/range-finding test was performed.

Post exposure observation period:

Immobility was recorded after 24 and 48 hours of exposure. Further clinical signs, e.g. daphnids trapped on the surface on the surface, were recorded at the same time.

Test conditions

Test temperature:

The temperature of the medium was continuously measured in a temperature control vessel, beginning at the start of the test.

The temperature of the test medium was between 19.8 and 20.1°C at the start of the test. The
temperature continuously measured in a temperature control vessel varied between 19.4 and
20.0
ºC during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

The pH was measured at the beginning of the test for all test solutions and the control. At the end of the test only measurements were performed in the control and the undiluted filtrate.

These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not
varying by more than 1.5 unit

Dissolved oxygen:

The dissolved oxygen concentration was measured at the beginning and at the end of the test, for the control and the undiluted filtrate.
These test conditions remained within the limits prescribed by the protocol; oxygen: >= 3 mg/L at the end of the test).

Salinity:

n/a

Nominal and measured concentrations:

Analysis of the samples taken from the filtrate prepared at 100 mg/L showed an estimated concentration of 4 and 7 μg/L at the start and at the end of the test, respectively. This resulted in an estimated average exposure concentration of 5 μg/L. On the filter used to prepare the test concentrations the test substance was identified, which is indicative of the use and removal of Proketal during the test solution preparation.

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass culture vessel
- fill volume: 5 litres
- Renewal rate of test solution (frequency/flow rate): renewed after 7 days of cultivation and half of the medium was renewed twice a week
- No. of organisms per vessel: approx 250

Composition of medium in table below

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 5 µg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Due to the very low solubility of Proketal in the test medium, concentration levels that might be toxic for daphnids could not be reached.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Under the conditions of the present study Proketal did not induce acute immobilisation of Daphnia magna at an estimated average concentration of 5 μg/L after 48 hours of exposure (NOEC).

Results with reference substance (positive control):

Calculation of EC50:
No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration).

Statistical analysis of the data was not needed as the effects recorded were not significant (<10%). Therefore, the NOEC was considered to be the highest concentration tested.

Acceptability of the test
1. In the control, no daphnids became immobilized.
2. The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

Reported statistics and error estimates:

Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).
Therefore, the NOEC was considered to be the highest concentration tested.

Determination of the average exposure concentrations: The average exposure concentrations were calculated as the geometric means of the concentrations of Proketal measured in the samples taken at the start (C t=0) and the end of the test (C t=48).

Any other information on results incl. tables

Table 1Acute immobilization of daphnids after 24 and 48 hours

 

Proketal % filtrate prepared at 100 mg/L	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 [1] 0 0 0	0
1.0	A B	5 5	0 0	0	0 0	0
10	A B	5 5	0 0	0	0 0	0
100 (5)	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0

[ ] number of daphnia observed trapped at the surface of the test solutions. This organism was reimmersed into the respective solution before the recording of mobility.

( ) measured concentration and expressed as the estimated average concentration (μg/L)

Table 2    Effectparameters

 

Parameter	Concentration Proketal (μg/L)1
NOEC	5
24 and48h-EC50	>5

1: Estimated average exposureconcentration

 

Table 3    pH and oxygenconcentrations

 

Proketal % filtrate preparedat 100mg/L	Start (t=0h)		End (t=48h)
	pH	O2	pH	O2
Control	8.0	9.5	8.0	9.2
1.0	8.0
10	8.0
100(5)	8.0	8.6	8.0	9.2

( ) measured concentration and expressed as the estimated average exposure concentration(μg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Proketal did not induce acute immobilisation of DapThis concentration of the undiluted filtrate was considered the maximum soluble concentration in the test medium.

The 48h-EC50 was above the range tested, i.e. exceeded an estimated average concentration of 5 μg/L.

Due to the very low solubility of Proketal in the test medium, concentration levels that might be toxic for daphnids could not be reached.