Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

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:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling schedule:
- Control: at 72 hours
- Test concentration at 0 and 72 hours

Vehicle:

no

Remarks:

auxiliaries: ultrasonic bath, magnetic stirrer, folded filter

Details on test solutions:

Only one test item concentration was prepared:
- 200.1 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer
- undissolved particles of the test item were removed by filtration(folded filter with a pore size of 7 - 12 μm)
- pH was measured to be 7.9

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus 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)
- 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)
- growth medium (according to BRINGMANN & KÜHN (1977)): renewed once a week
- cell density measurements: microcell counter, Sysmex F300, Digitana
- Preparation of pre cultures: Pre cultures were set up three days before the start of a test and were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium
- Final cell density: about 5000 cells per millilitre in the test medium.
- Growth medium and dilution water: Growth medium (OECD medium of OECD TG 201) was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

1.3 °dH

Test temperature:

21 - 24 °C

pH:

7.9 - 8.3

Nominal and measured concentrations:

100 mg/L (nominal);
1.04 mg/L (0 h) and 0.95 mg/L (72 h) (measured)

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: measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design: 1 test concentration plus 1 control, 6 replicates per concentration/control
- Initial cell density in the test cultures: approx. 5000 cells/mL; additionally highest test concentration without algae
- Test item concentration/s: 100 mg/L
- Method of administration: direct weighing
- Duration of exposure: 72 hours
- Criteria of effects: item-induced inhibition of yield [y] and growth rate of the algal population

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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.99 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.:

>= 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.99 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.:

> 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The nominal concentration was 100 mg/L for each dose descriptor.

Validity criteria fulfilled:

yes

Remarks:

cell density in the control cultures increased by a factor of at least 16 within 72 hours (found 118.0); mean coefficient of variation did not exceed 35 % (found 18.6%); coefficient of variation did not exceed 7 % (found 3.2%)

Conclusions:

No toxic effects of Macrolex Orange R against algae were observed at the limit of water solubility under exposure conditions after 72 h.

Executive summary:

The study was conducted as a static test 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).

Exponentially growing algal cells (Desmodesmus subspicatus; former name: Scenedesmus subspicatus) were exposed for 72 hours to a limit test concentration of nominally 100 mg/L of Macrolex Orange R dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

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 test item concentration was measured by HPLC. In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate, relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances procedure. After 72 h EC10, EC50, NOEC and LOEC were determined as > 0.99 mg/L (geometric mean measured concentration) referring to growth rate. No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

Description of key information

The study was conducted as a static test 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).

Exponentially growing algal cells (Desmodesmus subspicatus; former name: Scenedesmus subspicatus) were exposed for 72 hours to a limit test concentration of nominally 100 mg/L of Macrolex Orange R dissolved in dilution water. After 72 h EC10, EC50, NOEC and LOEC were determined as higher than 0.99 mg/L (geometric mean measured concentration) referring to growth rate. No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.99 mg/L

EC10 or NOEC for freshwater algae:

0.99 mg/L

Additional information

"Should be read as >0.99 mg/L."