3'H-Cycloprop[15,16]androst-15-en-17-one, 3-(2,2-dimethyl-1-oxopropoxy)-5,6-epoxy-15,16-dihydro-7-hydroxy-, (3.beta.,5.beta.,6.beta.,7.beta.,15.alpha.,16.alpha.)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul - Sep 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5,6 beta-Epoxy-7 beta-hydroxy-15 beta,16 beta-methylene-3 beta-pivaloyloxy-5 beta-androstan-17-one

EC Number:

617-351-6

Cas Number:

82544-13-6

Molecular formula:

C25 H36 O5

IUPAC Name:

5,6 beta-Epoxy-7 beta-hydroxy-15 beta,16 beta-methylene-3 beta-pivaloyloxy-5 beta-androstan-17-one

Details on test material:

- Name of test material (as cited in study report): Methylenepoxidpivalat (ZK 90346
- Analytical purity: 92.1%
- Lot/batch No.: 21607513

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the stock solution 100 mg of ZK 90346 were suspended in 1000 mL demineralized water under constant stirring for approximately 24 hours at room temperature after 30 min. ultrasonification. This is the highest concentration to be tested according to the EC guideline. Thereafter, the suspension was filtered through a glassfibre filter (0.2 µm).
- Eluate: no
- Differential loading: yes

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): "Sammlung von Algenkulturen, Universität Göttingen", and kept on slant agar containing a nutrient solution under standardized conditions (10-15 °C, diffuse light [light/dark rhythm 12h/12h]).
- Age of inoculum (at test initiation): pre-culture was incubated for 3 days

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 25 °C

pH:

The pH-value in the stock solution was pH=9.1

Nominal and measured concentrations:

Nominal: 10 mg/L
Measured: 7.2 mg/L (saturated solution)

Details on test conditions:

TEST SYSTEM
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: approx. 6000 - 8000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Fluorescence measurement (wavelength of 320 and 800 nm for agitation and measurement, respectively)
- Other: The algae were microscopically examined at the end of exposure.

CULTURING APPARATUS
-Details on culturing apparatus used: Abimed AlgenTest XT

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No changes in morphological structures or other abnormalities were observed.

Reported statistics and error estimates:

The values for the average area below the growth curves, specific growth rate, and the percentage inhibition were calculated by the computer program Abimed "AlgenTest XT 2.21". The program calculates the respective parameters according to the mathematical formulas given in the OECD guideline. The EbC50 (72 h) (biomass) and the ErC50 (72 h) (growth rate) as well as the 95% confidence limits are also calculated by the above referenced computer program on the basis of a probit analysis.

Any other information on results incl. tables

Inhibition of biomass and the growth rate (%) of Scenedesmus subspicatus after 72 hours exposure to ZK 90346 (negative values represent growth stimulation

	Concentration of	Inhibition in %
Dilution of ZK 90346	ZK 90346 (mg/L)	Biomass (integral)	Growth rate
Control	0,0 (control)	--	--
1:100	0,1	-13,6	-11,8
1:50	0,2	-15,6	-13,3
1:25	0,4	-15,5	-12,8
1:5	2	75,9	50,2
1:2,5	4	80,2	55,7
Saturated solution	8	88,6	67,2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72 h): 2.1 mg/L

Executive summary:

The purpose of this study was to determine the effects of the test compound Methylenepoxidpivalat (ZK 90346) on the growth of the green algae Scenedesmus subspicatus. ZK 90346 is an intermediate of the synthesis of Drospirenone. The study was conducted in agreement with the test guideline OECD no. 201.

The test substance ZK 90346 was incubated in an aqueous solution including nutrients with an algae population of Scenedesmus subspicatus for a test duration of approximately 72 hours in an electronically controlled dosing and incubation apparatus. For the preparation of the test solutions a suspension with a nominal loading of 125 mg/L ZK 90346 in water was stirred for 24 hours after 30 min of ultrasonification. This suspension was filtered through a glassfibre filter. The resulting solution served as the highest concentration (saturated solution). It was further diluted 1:2.5, 1:5, 1:25, 1:50, and 1:100 with demineralized water. Additionally, a control solution was prepared with demineralized water without test material.

ZK 90346 had a concentration-depended inhibitory effect on the growth of Scenedesmus subspicatus at dilutions of 1:5 and lower of a saturated solution. The EbC50 (biomass) was 0,3 mg/L [95% confidence limits: not calculated], the ErC50 (growth rate) was 2.1 mg/L [95% confidence limits: not calculated). Consequently, ZK 90346 was considered to be very toxic to the green algae Scenedesmus subspicatus, since the EbC50 was below 1 mg/L.