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

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Carried out under GLP conditions and per OECD method.

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 211 (Daphnia magna Reproduction Test)

Deviations:

no

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 C25H36O4
- Substance type:crystalline powder
- 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-ventilated place at room temperature (15-25 °C)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No reference substance

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: UPLC
- Sample storage conditions before analysis: The samples were refrigerated at 5±3°C after pretreatment if the samples were not analyzed within 24h


The concentration stability of the saturated solution was analyzed by UPLC at 0h, 24h, 48h and 72h.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
based on results of a range finding test, 6 treatment groups(3.13%, 6.25%, 12.5%, 25%, 50% and 100% of the saturated solution) and a control group were included in the test. A nominal concentration of 10mg/L of the test substance was prepared and stirred (via magnetic stirrer) in the dark for 72h at 500rpm followed by filtration using a 0.45µm nitrocellulose membrane to make a saturated solution of the test substance in the test water as the stock solution (regarded as "saturated solution" in this report, which arithmetic mean measured concentration was 20.0µg/L in the definitive test). Other test solutions were prepared by dilution of the saturated solution with Elendt M4 by using the vessels calibrated by measurement Institutes.

(Elendt M4 medium was prepared by analytical reagent and deionized water. The test medium was aerated for 30 minutes prior to the start of the test and placed in testing temperature at least 12 hours before the test. At the beginning and during the test, the dissolved oxygen concentration was 8.11-8.?0mg/L; the pH was 6.45-6.90; the hardness was 150-160mg/L(asCaC03). The concentration of TOC in the medium (i.e. before addition of the algae) was less than 0.4mg/L(LOQ of TOC method was 0.8 mg/L). The ingredients and their nominal concentrations of Elendt M4 Medium are attached below.)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Age of parental stock (mean and range, SD): Young female Daphnia (the parent animals) were aged less than 24 hours and not first brood progeny were used at the start of the test
- Food type: unicellular green algae Desmodesmus Subspicatus and Pseudokirchneriella subcapitata
- Amount: rate equivalent to 0.1- 0.2mg of organic carbon per adult per day

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Daily

Test conditions

Hardness:

150-160mg/L (asCaCO3) -definitive test
hardness of the test solutions ranged from 155 to 165mg/L(CaC03). range finding

Test temperature:

water temperature: 20.1 -21.2°C
During the test period, the temperature in the incubator were between 20.1 and 20.2°C.(range finding)

pH:

the pH values of the test solutions ranged from 7.830 to 8.374, range finding
the pH values of the test solutions ranged from 6.32 to 6.90, definitive test

Dissolved oxygen:

Dissolved oxygen concentrations of the test solutions were no less than 8.11-8.70mg/L.
Oxygen concentrations of the test solutions were no less than 3mg/L (8.18-9.76mg/L), (range finding)

Salinity:

n/a

Nominal and measured concentrations:

Nominal concentrations of the treatment group were 3.13%, 6.25%, 12.5%, 25%, 50% and 100% saturated solution. The mean measured concentration of the saturated solution was 20.0 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 75ml glass container
- Material, size, headspace, fill volume:
- Aeration: artifical climate chamber
- Renewal rate of test solution : The frequency of medium renewal was three times per week. The medium was renewed on days 2, 4, 7, 9, 11, 14, 16,
18.
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): six

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH:pH: 6.45-6.90;
- Photoperiod: Light : Dark=16 hours : 8 hours;
- Light intensity: 1112-13921ux;

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Oxygen concentration, temperature, hardness and pH values were measured twice per week, in fresh and old medium, in the control(s) and in the highest test substance concentration.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Four treatment groups and one test-medium control series were included in the range-finding test. The nominal concentrations of the treatment group were 0.1 %, 1 %, 12.5%, 10% and 100% saturated solution.
- Results used to determine the conditions for the definitive study: The mortality of the parent animals in the control group and the treatment groups were 0%. According to the result, the reproduction inhibition percentage of the 100% saturated solution to Daphnia magna was 48.6% and the reproduction inhibition percentage of the 0.1 % saturated solution to Daphnia magna was -14.72%.
Based on the result of range-finding test, a definitive test was carried out.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

23.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limit was 10.8-422µg/L

Details on results:

- Mortality of parent animals: 0%
- No. of offspring produced per day per female: Please see table attached below "number of live offspring and the coefficient of variation for control fecundity"
- Other biological observations:
No aborted eggs and dead offspring were observed during the test. According to the result of the Maximum Likelihood-Probit Test, the 21 days NOEC for reproduction of the test substance to Daphnia magna was 2.50µg/L and the LOEC was 5.00µg/L. The 50 % reduction in reproductive output(EC50) was 23.8µg/L(95% confidence limit was 10.8- 422µg/L). During the test period, the temperature in the incubator were between 20.1 and
21.2°C, the pH values of the test solutions ranged from 6.32 to 6.90, the dissolved oxygen concentrations of the test solutions were no less than 3mg/L(8.10-8. 70mg/L), and the hardness of the test solutions ranged from 150 to 160mg/L(CaC03) (See table attached below "number of live offspring and the coefficient of variation for control fecundity"


The concentration stability of the saturated solution was analyzed by UPLC at 0h, 24h, 48h and 72h. The result showed that, compared with the concentration at 0h, the variation range were 2.21-19.9%, all were within +20% of the initial concentration

The concentration of the saturated solution was proved to be stable within 72h. Therefore, the arithmetic mean measured concentration of 20.0µg/L for all 100% saturated solutions were used as the measured concentration of 100% saturated solution in the definitive test.

Reported statistics and error estimates:

Statistical analysis of the data was accomplished withToxcalc v5.0.32.  In addition, and as far as possible, the data are analysed using a regression model in order to estimate the concentration that would cause a x% reduction in reproductive output,

A plot of total number of living offspring produced per parent animal in each replicate
excluding any parent animal which may have accidentally or inadvertently died during
the test vs. concentration of the test substance was achieved. For the estimation of
the concentration which would cause a 50% reduction in reproductive output(i.e. the
ECso) and 95% confidence limits.

Any other information on results incl. tables

The mortality of the parent animals in control group was 0% at the end of the test;

 The accidental or inadvertent mortalities of the parent animals in each treatment group were 0%;

 The mean number of live offspring produced per parent animal surviving at the end of the test in control group was 88.

 The test is valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Expressed as the series of concentrations calculated by the measured mean concentration of the saturated solution of the test substance, the lethal effect of Proketal (NJBY20130218-0718) on Daphnia magna is as following:

The 21 days NOEC value was 2.50µg/L;
The 21 days LOEC value was 5.00µg/L;
The 21 days ECso value was 23.8µg/L(95% confidence limits was 10.8-422µg/L).