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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Sampling schedule:
Control: at 72 hours
Test concentration/s: 10 mg/L and 100 mg/L at 0 and 72 hours
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and preparation of test item concentrations
A stock solution was prepared to give the desired series of test concentrations. 200.1 mg of the test item were added to 2 litres of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and stirred for 24 h on a magnetic stirrer. The pH was measured to be 7.9.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 1.058 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.
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) 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 particle counter, Z2, Beckmann Coulter.
- 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 (annex 1).
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 24 °C
pH:
7.8 - 8.2
Nominal and measured concentrations:
nominal: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Effective concentrations ranged from 98.9 % to 122 % of nominal values at 0 hours, and from 98.0 % to 107 % of nominal values at 72 hours.
Details on test 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. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a particle counter (Z2, Beckmann Coulter) by taking aliquots from each test flask, which were not replaced.
- Experimental design: 8 concentrations plus 1 control, 3 replicates per concentration, 3 replicates per control, additionally highest test concentration without algae
- Initial cell density in the test cultures approximately 5000 cells per millilitre
- Test item concentration/s: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
- Method of administration: stock solution (100 mg/L)
- Duration of exposure: 72 hours
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 98.9 % to 122 % of nominal values at 0 hours, and from 98.0 % to 107 % of nominal values at 72 hours.
Reported statistics and error estimates:
The 72 hour EC 10 and EC 50 were calculated by probit analysis.
All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 100000.
Validity criteria fulfilled:
yes
Remarks:
cell density in controls increased by a factor >= 16 in 72 h; mean coefficient of variation (CV) for section-by-section specific growth rates in controls <= 35%, CV of average specific growth rates during the whole test period in replicate control <= 7%
Conclusions:
A 72h-EC10 of 9.2 mg/L and a 72h-EC50 of 26 mg/L were determined for the test substance towards algae under exposure conditions.
Executive summary:

A study was performed to assess the adverse effects of the test substance on the growth rate of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations 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. The following nominal concentrations of the test item were tested in the range finding test: 0.1, 1.0, 10 and 100 mg/L.

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of test substance dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax and a magnetic stirrer.

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.

In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), 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 Williams Multiple Sequential t-test Procedure. The following values were determined after 72 h: EC10 = 9.2 mg/L, EC50 = 26 mg/L.

The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 98.9 % to 122 % of nominal values at 0 hours, and from 98.0 % to 107 % of nominal values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of the test substance on the growth rate of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations 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. The following nominal concentrations of the test item were tested in the range finding test: 0.1, 1.0, 10 and 100 mg/L.

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of test substance dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax and a magnetic stirrer.

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.

In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), 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 Williams Multiple Sequential t-test Procedure. The following values were determined after 72 h: EC10 = 9.2 mg/L, EC50 = 26 mg/L.

The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 98.9 % to 122 % of nominal values at 0 hours, and from 98.0 % to 107 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:
26 mg/L
EC10 or NOEC for freshwater algae:
9.2 mg/L

Additional information