9beta,11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

pH value increased by more than 1.5 pH units, this is not regarded to be relevant to the results as all validity criteria were met

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

pH value increased by more than 1.5 pH units, this is not regarded to be relevant to the results as all validity criteria were met

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC analysis, UV/VIS-detection

Details on sampling:

Control at 72 h
Test concentrations at 0 h, 24 h, 48 h, 72 h

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations
- 200.7 mg of test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 1 h on a magnetic stirrer
- undissolved particles of test item were removed by filtration using a folded filter (pore size: 7 - 12 μm)
- pH was measured to be 7.8
- To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany)
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977)) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: algal inocula for the test were taken from an exponentially growing pre culture and mixed with the growth medium
- Final cell density: about 5000 cells per millilitre in the test medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21 - 24 °C

pH:

7.9 - 10.1

Nominal and measured concentrations:

Nominal [mg/L]: 6.3, 12.5, 25, 50, 100
Effective concentrations ranged from 1.2 % to 2.5 % of nominal values at 0 hours, from 0.5 to 1.2 % of nominal values at 24 hours, from 0.2 % to 0.5 % of nominal values at 48 hours and correspond to 0.3 % of nominal values at 72 hours.

Details on test conditions:

Exposure conditions
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
- Light intensity: A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 5 test concentrations plus 1 control, 3 replicates per concentration, 3 replicates per control
- Initial cell density in the test cultures: approximately 5000 cells per millilitre
- additionally highest test concentration without algae
- Test item concentrations: 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration: stock solution
- Duration of exposure: 72 hours
- Criteria of effects: item-induced inhibition of yield [y] and growth rate [r] of the algal population

Calculations:
- Statistics programme: ToxRatPro Version 2.10 (released 2010-09-10)
- For calculations all algae counts were divided by a factor of 10000

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 1.2 % to 2.5 % of nominal values at 0 hours, from 0.5 to 1.2 % of nominal values at 24 hours, from 0.2 % to 0.5 % of nominal values at 48 hours and correspond to 0.3 % of nominal values at 72 hours.

The pH value increased by more than 1.5 pH units. This increase is not regarded to be relevant to the results as all validity criteria were met.

Validity criteria fulfilled:

yes

Remarks:

increase of cell density in the control cultures >= 16 within 72 h; mean coefficient of variation for section-by-section specific growth rates <= 35 %; coefficient of variation of average specific growth rates <= 7 %

Conclusions:

After 72 h no toxic effect towards algae could be found for Epoxid-Valerat up to the highest concentration and up to water solubility.

Executive summary:

To assess the adverse effects of Epoxid-Valerat on the growth rate of algae Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations a study was performed in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test.

In the main test, the algae were exposed to a range of concentrations of Epoxid-Valerat (nominally 6.3, 12.5, 25, 50 and 100 mg/L) for a period of 72 hours. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter. Cell densities were recorded at 24 hour intervals. The 72 hour EC 10 and EC 50 were calculated by probit analysis. During the test a temperature range of 21 - 24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure. The maintenance of the test item concentrations was proved by analytical measurements. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, at all measuring points (0, 24, 48 and 72 hours) a test flask without algae was run in parallel to the geometric series of test concentrations.

After 72 hours of exposure the following values were determined [mg/L]: ErC50 > 0.27, ErC10 > 0.27, NOEC ≥ 0.27, LOEC > 0.27. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 1.2 % to 2.5 % of nominal values at 0 hours, from 0.5 to 1.2 % of nominal values at 24 hours, from 0.2 % to 0.5 % of nominal values at 48 hours and correspond to 0.3 % of nominal values at 72 hours.

Description of key information

After 72 h no toxic effect towards algae could be seen for Epoxid-Valerat up to the highest concentration and up to water solubility. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 1.2 to 2.5 % of nominal values at 0 hours, from 0.5 to 1.2 % of nominal values at 24 hours, from 0.2 to 0.5 % of nominal values at 48 hours and corresponds to 0.3 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.27 mg/L

EC10 or NOEC for freshwater algae:

0.27 mg/L

Additional information

"Should read: 72 h-EC50 >0.27 mg/L and 72 h-NOEC >0.27 mg/L"