Estr-5(10)-en-17-one, 3,3-dimethoxy-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

not relevant

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

As a parameter for the cell density, the chlorophyll fluorescence in the test and control solutions was determined after 24,48 and 72 hours of incubation

Vehicle:

no

Details on test solutions:

I. Preparation of the stock solution:
- 100 mg of test material was suspended in 1000 mL demineralized water with the aid of ultrasonification (30 min.) under constant stirring tor approximately 24 hours
- Thereafter, the suspension was filtered through a glassfibre filter (0.2 µm)

II. Preparation of the test solutions:
- The test concentrations were selected according to a preceding test, where a saturated solution and dilutions up to 1:100 showed almost complete growth inhibition
- Therefore, the filtered saturated solution was further diluted 1:80 to prepare the stock solution
- An electronically controlled syringe prepared the required test concentrations by diluting aliquots of the stock solution 1:1.25, 1:200, 1:400, 1:800, 1:1600, and 1:3200 with demineralized water in order to prepare the appropriate test concentrations
- Further, a control prepared with demineralized water was set up

III. Addition of inoculum:
- 10 mL of nutrient solution were added to the 80 mL test substance solutions
- Finally, 10 mL of the inoculum were added, all by electronically controlled syringing
- Each test concentration was set up in triplicate, the control without test compound was incubated in six paralleis

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

I. Culturing of the test organism and preparation of inoculum:
- The alga was obtained trom the "Sammlung von Algenkulturen, Universität Göttingen" (Germany), and kept on slant agar eontaining a nutrient solution at 10-15 °C under light/dark rhythm (12h/12h in diffuse light)
- This culture was dissolved in a liquid nutrient solution and subsequently transterred approximately once weekly

II. Preparation of the pre-culture:
- The algae tor the pre-culture were taken from a stock up to seven days old, approx. 96 hours before the start of the incubation in the test
- They were kept in nutrient solution starting with an approx. cell density of 10 000 cells/mL, counted with the Coulter Counter
- This pre-culture was incubated for 4 days in an incubator shaker apparatus
- For technical reasons the incubation was extended trom the recommended period of 72 hours, which had no effect on the viability and condition of the inoculum
- The temperature was held at approx. 24°C
- After the incubation period the cell concentration of the algae reached a sufficient density (approx.1.2 Mill. cells/mL), and the pre-culture was diluted to 100000 cells per mL with demineralized water, ready to be used as inoculum

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

23 - 24 °C (during the whole test period)

pH:

pH at the end of the test: 8.5 and 10.4

Dissolved oxygen:

No data

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

The resulting solution of 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200 with corresponds to the test concentration of 0.006, 0.013, 0.025, 0.05. 0.1 and 0.2 mg/L test concentration calculated on the basis of TOC analysis

Details on test conditions:

I. Incubation conditions:
- The test vessels were incubated under intermittent stirring for approximately 72 hours in an incubating apparatus (Abimed AlgenTest XT) with continuous light (at approximately 6000-8000 lux) and controlled temperature (water bath)

II. pH and temperature measurements:
- The pH-value was measured in the stock solution in order to assess appropriate conditions
- After the exposure the pH was measured in two control and one test vessel of each concentration
- The temperature in the water bath was continuously monitored by a minimax thermometer

III. Fluorescence determination:
- All measurements were made by fluorescence photometer at a wavelength of 320-800 nm after 24, 48 and 72 hours
- Sampling volume and sampling time were electronically controlled
- For statistical reasons, it was aimed that the coefficient of variation between replicates at 72 hours did not exceed 25 %

IV. Microscopic examination:
- The algae were microscopically examined at the end of exposure
- The algae of one test vessel of each test concentration group were analyzed together with two of the control algae with regard to cell abnormalities

V. Calculation 01 results and statistical analysis:
- 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

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limit could not be calculated

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limit 0.08-0.14 mg/L

The growth (biomass and growth rate) increased at the three lowest concentrations, while it was

inhibited at the three highest concentrations. There was a clear concentration related inhibitory ,

effect up to 88% and 82% for biomass and growth rate, respectively.

The organic carbon concentration of a saturated solution was analyzed with a TOC during an acute immobilization Daphnia magna test (Schering report A00488). The substance concentration was calculated to be approximately 20 mg/L. The concentrations during this algal test were estimated according to this analysis.

Validity criteria fulfilled:

yes

Conclusions:

Dimethoxyketal is very toxic to the green algae Scenedesmus subspicatus, since the EC50 was markedly below 1 mg/L.

Executive summary:

The purpose of this study was to determine the effects of the test compound Dimethoxyketal (ZK 235382) on the growth of the green algae Scenedesmus subspicatus. Dimethoxyketal is an intermediate of the synthesis of dienogest. The study was conducted in agreement with the test guideline OECD No. 201. The 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 nutrient solution was made up of mainly nitrate, phosphates and some trace elements. For the preparation of the test solutions a suspension with a nominal loading of 100 mg/L test substance was stirred for 24 hours after ultrasonification. This suspension was filtered through a glassfibre filter. The resulting solution was further diluted 1:100, 1 :200, 1 :400, 1 :800, 1 :1600,

and 1 :3200 with demineralized water, representing the test solutions. Additionally, a control solution was prepared with demineralized water without test material. As a parameter for the growth of the algae population, the fluorescence of the algal cells was

measured with a fluorescence-photometer. The increase of biomass and the growth rate were calculated on the basis of the fluorescence. The calculated biomass and growth rate of each concentration were compared to those of the

controls, and the inhibition was calculated.

Dmethoxyketal had a concentration-dependent inhibitory effect on the growth of Scenedesmus subspicatus at the higher concentration tested. The EbC50 (biomass) was 0.06 mg/L [95% confidence limits could not be calculated due to the concentration-effect relationship], the ErC50 (growth rate) was 0.10 mg/L [95% confidence limits: 0.08-0.14] on the basis of the estimated concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.1 mg/L

Additional information

Value based on growth rate.