3'H-Cycloprop[15,16]androsta-6,15-dien-17-one, 15,16-dihydro-3,5-dihydroxy-, (3.beta.,5.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:

Sept 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

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:

Reference substance 001

Cas Number:

82543-15-5

Test material form:

solid

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 38700 were suspended in 1000 mL demineralized water, treated in an ultrasonic bath for 30 min, and stirred for approximately 24 hours at room temperature. This is the highest concentration to be tested according to the EC guideline (2). Thereafter, the suspension was filtered through a glassfibre filter (0.2 µm). The filtered stock solution was used for the highest test concentration (equivalent to "saturated solution"), which is a 1: 1.25 dilution by adding nutrient solution and the inoculum. The "saturated solution" was further diluted with demineralized water by an electronically controlled syringe resulting in 1:2.5,1:5,1:10,1:25 and 1:50 dilutions of the stock solution.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

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): The alga was obtained from the "Sammlung von Algenkulturen, Universität Göttingen"
- Method of cultivation: kept on slant agar containing a nutrient solution under standardized conditions (10-15 °C, diffuse light [light/dark rhythm 12h/12h)). This culture was dissolved in a liquid nutrient solution and subsequently transferred approximately once weekly. The algae for the pre-culture were taken from a stock up to seven days old, approximately 96 hours before the start of the incubation in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 °C (± 2)

pH:

9.3 - 9.7

Nominal and measured concentrations:

Measured: 1.2, 2.4, 5.9, 11.7, 23.5, 47.0 mg/L

Details on test conditions:

TEST SYSTEM
- Initial cells density: approx. 10000 cells/mL
- Control end cell density: approx. 1053200 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 lighting
- Light intensity and quality: approx. 6000 - 8000 lux

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

CULTURING APPARATUS
-Details on culturing apparatus used: Controlled temperature water bath

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 47 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.:

> 47 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
- Unusual cell shape: In the dilutions up to 1:5 no changes in morphological structures or other abnormalities were observed. In the 1:2.5 dilution the size of cells were partially decreased abnormality where in the highest concentration (1: 1.25 dilution equivalent to "saturated solution") cell damage were noted.

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 (1). The EbC50/72 hours (biomass) and the ErC50 72 hours (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 Endiol (ZK 38700)

		Inhibition in %
Dilution	Concentration of Endiol (mg/L)	Biomass	Growth rate
0	0.0 (control)	--	--
1:50	1.2	-4.1	0.2
1:25	2.4	8.8	4.5
1 :10	5.9	14.0	7,3
1:5	11.7	19.4	6.9
1:2.5	23.5	27.0	7.3
Saturated solution	47.0	43.9	17.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Endiol was considered not to be toxic to the green algae Scenedesmus subspicatus, since the EC50 was higher than its solubility.

Executive summary:

The purpose of this study was to determine the effects of the test compound Endiol (ZK 38700) on the growth of the green algae Scenedesmus subspicatus. Endiol is an intermediate of the synthesis of Drospirenone. The study was conducted in agreement with the test guideline OECD no. 201 and the EC guideline Part 2 - Testing Methods, Part C.3.

The test substance 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.

The organic carbon concentration of the stock solution was analyzed with a TOC analyzer at the start of the incubation. The substance concentration was calculated on the basis of the molecular formula. The highest test concentration (equivalent to "saturated solution") was 47.0 mg/L.

The biomass and growth rate was inhibited (> 10%) from concentrations of 5.9 mg/L and higher. This was considered to be a compound related effect. Endiol had a concentration-depended inhibitory effect on the growth of Scenedesmus subspicatus. The EC50 for biomass and growth rate, however, was above the solubility. Consequently, Endiol was considered not to be toxic to the green algae Scenedesmus subspicatus, since the EC50 was higher than its solubility.