Pregn-4-ene-3,20-dione, 21-(acetyloxy)-6-fluoro-11-hydroxy-16-methyl-, (6.alpha.,11.beta.,16.alpha.)-

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:

Nov 1998 - Jul 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

TOC analyser

Details on sampling:

The organic carbon concentration of the stock solution was analyzed in a single determination with a TOC analyzer before the start of the incubation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the preparation of the test solutions a suspension with a nominal loading of 102 mg/L test item was stirred for approximately 24 hours. This suspension was filtered through a glassfibre filter. The resulting solution served as the highest concentration (saturated solution). It was further diluted 1:10, 1:100 and 1:1000 with demineralized water.
- Eluate: no
- Differential loading: no
- Controls: yes, test meidum control

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM:
- Source: "Sammlung von Algenkulturen, Universität Goettingen" (Collection of Algae Cultures, University Goettingen, Germany)
- Maintenance and Acclimatisation: kept on slant agar containing a nutrient solution under standardized conditions (10-15 °C, diffuse light (light/dark rhythm 12 h/12 h)); this culture was dissolved in a liquid nutrient solution and subsequently transferred approx. once weekly

Preparation of pre-cultures
- algae for the pre-culture were taken from a stock up to seven days old, approx. 72 hours before the start of the incubation in the test; kept in the concentrated nutrient solution (diluted 1:10), starting with an approximate cell density of 10,000 cells/mL, counted with the Coulter Counter
- Incubation conditions: incubated for 3 days under continuous light in an incubator shaker apparatus; the temperature at approx. 23 °C
- Test cultures: After the incubation period the cell concentration of the algae reached a sufficient density (approx. 1 167 200 cells/mL), and the pre-culture was diluted to approx.100,000 cells/mL with mineralized water, ready to be used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 °C

pH:

at the beginning of the test: 8.0 - 8.3
at the end of the test: 8.4 - 8.7

Nominal and measured concentrations:

- TOC analysis of the stock solution gave a value of 2.133 mg TOC/L corresponding to 2.603 mg/L test item (further diluted 1: 1 0, 1: 1 00 and 1: 1000 with demineralized water)
- nominal: 2.603, 0.260, 0.026, 0.002 mg/L

Details on test conditions:

TEST SYSTEM
- Control end cells density: 667 cells/mL x 10E+03
- 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
- Intervals of water quality measurement: Temperature in the incubator was recorded at the beginning and at the end of incubation. pH-value was measured in all test vessels at the beginning and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: continuous light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter every 24 h
- Microscopic examination: Algae were microscopically examined at the end of exposure. One test vessel of each test concentration group was compared with selected control algae with regard to cell abnormalities.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: on the basis of TOC analysis

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.603 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: saturated solution

Details on results:

There was a slight reduction in growth at the 1:100 and 1:10 dilutions. However, the saturated solution did not show growth inhibition. Hence, this slight growth reduction was not considered substance-related but due to biological variability.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No relevant observations were made in comparison with the algae of the control group.

Reported statistics and error estimates:

The values for the average specific growth rate, the area below the growth curves, the percentage inhibition of the cell growth and the reduction in average growth rate were calculated by the computer program "ALG" (version 1.2), Informatik CMF, Schering AG, Berlin, Germany.

Validity criteria for the measurement of the algae toxicity

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
Exponentially growing test organisms are exposed to the test substance in batch cultures over a period of normally 72 hours.	Test organisms: Scenedesmus subspicatus Test period: 72 h	Yes
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Growth in the control flasks after 3 days showed a reproduction rate that was greater than a factor of 16. Areas ("biomass integrals") under the growth curves of Scenedesmus subspicatus also showed a minimum of 16-fold increase after 72 h.	Yes
For the final definitive test at least five concentrations, arranged in a geometric series with a factor not exceeding 3.2, should be selected. For test substances showing a flat concentration response curve a higher factor may be justified. The concentration series should preferably cover the range causing 5-75 % inhibition of algal growth rate.	Test concentration no.: 5 Factor of geometric series of test conc.: 10	Yes with restrictions
The cultures should be maintained at a temperature in the range of 21 to 24°C, controlled at ± 2°C.	Incubation was at 23 ± 0°C	Yes
The pH of the control medium should not increase by more than 1.5 units during the test.	Solvent control medium pH was 8.0 at the beginning of the test and 8.7 after 72 h.	Yes

 

Validity criteria fulfilled:

yes

Remarks:

see "Any other information on results incl. tables"

Conclusions:

ErC50 (72 h) > 2.603 mg/L (nominal)

Executive summary:

The purpose of this study was to determine the effects of Fluocortolon-A-Acetat on the growth of the green algae Desmodesmus subspicatus (former name: Scenedesmus subspicatus). The study was conducted in agreement with the test guideline OECD no. 201 and EC guideline C.3.

Fluocortolon-A-Acetat was incubated with an algae population of Scenedesmus subspicatus for a test duration of approximately 72 hours. For the preparation of the test solutions a suspension with a nominal loading of 102 mg/L test item was stirred for approximately 24 hours. This suspension was filtered and the resulting solution served as the highest concentration (saturated solution). The organic carbon concentration of the stock solution was analysed with a TOC analyzer at the start of the incubation as 2.6 mg/L. It was further diluted 1:10, 1:100 and 1:1000 with demineralized water to give nominal concentrations of 0.26 mg/L, 0.026 mg/L and 0.002 mg/L. The algae were exposed to each concentration in triplicate. Six vessels were prepared for the control. The algae were incubated in an incubator shaker under continuous light and a temperature of approximately 23 °C. The pH-values were measured at the beginning and end of exposure. As a parameter for the growth of the algae population, the cell concentrations of the test and control solutions were counted with an electronic particle counter ("Coulter Counter").

The 72h-EC50 of Fluocortolone-A-Acetat for green algae Desmodesmus subspicatus was higher than a saturated solution (2.6 mg/L). Therefore Fluocortolone-A-acetate is not toxic to the green algae Desmodesmus subspicatus.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algae study.

Description of key information

A study was conducted in agreement with the test guideline OECD no. 201 and EC guideline C.3 to determine the toxicity of Fluocortolon-A-Acetat towards algae. The 72 h-ErC50 of Fluocortolone-A-Acetat for green algae Desmodesmus subspicatus was higher than a saturated solution (ErC50 > 2.603 mg/L (nominal)).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.603 mg/L

Additional information

"Should read: >2.603 mg/L"

There was a slight reduction in growth at the 1:100 and 1:10 dilutions. However, the saturated solution (2.603 mg/L) did not show growth inhibition. Hence, this slight growth reduction was not considered substance-related but due to biological variability.