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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Feb - 17 Jul 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.3 (Algal Inhibition test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Internal standard solution:
30.0 mg Cyclododecane were dissolved in methanol and made up with methanol to the mark in a 20 ml volumetric flask to prepare a solution of 1500 µg/L. This solution is used as internal standard solution.

Standard solutions:
50.0 mg of the analytical standard were dissolved in acetonitrile and made up with acetonitrile to the mark in a 25 ml volumetric flask (2000 mg/L). 1 ml of this solution was diluted to 20 ml with acetonitrile to prepare a stock solution of 100 mg/L. Defined volumes of this stock solution and an aliquot of the internal standard solution were added to 15 ml water, 2 pellets potassium hydroxide and about 4.5 g sodium sulfate resulting in standard solutions in a range of 0.05 to 12.0 mg test item /I water.
These mixtures were extracted with 4 ml diethylether. The organic extract was used to calibrate the GC-system.
Although the overall method sensitivity may be better, the concentration of the lowest used calibration solution is employed as the limit of quantification.

Sample storage conditions before analysis:
Routinely, the samples are analysed immediately. Only in exceptional cases, they are stored overnight deeply frozen and protected from light.
Details on test solutions:
A direct weighing was prepared to give the desired test concentration. To achieve this 125.3 mg of the test item were added to 1 litre of dilution water and treated with an ultra turrax for 60 seconds at 8000 rpm and afterwards stirred for 1 hour on a magnetic stirrer.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Source: on-axenic strain of the test species obtained from The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingen (Germany).
Maintenance of stock cultures: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions(23 +/- 2°C) at a light intensity in the range 60 - 120jiE. x m"2 x s"1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KUHN (1977)is renewed once a week. Cell density measurements are made using a microcell counter.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
None reported.
Test temperature:
None reported.
Nominal and measured concentrations:
nominal: 100 mg/L
meassured: 87.4 - 92.1 mg/L (0 h), 90.9 - 93.9 mg/L (72 h)
Details on test conditions:
Test vessels: 300 ml Erlenmeyer flasks with cotton stoppers
Culturing apparatus: Light chamber in which a temperature in the range 21°C to 25°C can be maintained at +/- 2°C, and continuous uniform illumination is provided in the spectral range 400 to 700 nm.
Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 1x, is recommended to use.
Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber.

Experimental design: 1 test concentration plus 1 control; 3 replicates per concentration, 6 replicates per control; initial cell density in the test cultures approximately 5000 cells per millilitre; additionally highest test concentration without algae

Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of growth [b] and growth rate [r], respectively, of the algal population.
Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
All results are expressed in terms of nominal concentrations. Recovery rates ranged from 87.4 - 92.1% of nominal values at 0 hours, and from 90.9 - 93.9% of nominal values at 72 hours, respectively.
Results with reference substance (positive control):

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: Desmodesmus subspicatus

Test period: 72 h

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. Cell density in the control flasks after 3 days showed a reproduction rate that was greater than a factor of 16 (factor 81).Yes
The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.In this specific study, the observed value is 18.6%.Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%.It was 2.05% in this study.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.: limit test (100 mg/L).

Not applicable
The cultures should be maintained at a temperature in the range of 21 to 24°C, controlled at ± 2°C.21°C to 25°C can be maintained at +/- 2°C.Yes (a small deviation of 1 °C is acceptable)
The pH of the control medium should not increase by more than 1.5 units during the test.pH in the control medium after 0 and 72 h was 8.1 - 8.6, respectively.



Validity criteria fulfilled:
See 'Any other information on results incl. tables'.
An 72h EC50 of > 100 mg/L was determined for the test substance, as no toxic effects against algae were observed.
Executive summary:

No toxic effects against algae at a concentration of 100 mg/l.

Description of key information

An 72h EC50 of > 100 mg/L was determined for the test substance, as no toxic effects against algae were observed.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

Should read > 100 mg/L.