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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:

23 April 2007 to 31 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.32, 1.0, 3.2, 10, 32, and 100 mg/L

For the characterization of the WAFs and the determination of the actual test item
concentrations the following samples were taken:

Just before the start of the test:
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)

After 72 hours:
- duplicate samples from each test medium (with algae)
(stability samples) - duplicate samples from the control (with algae)

For the 72-hour stability samples, additional flasks with algae were incubated at each
concentration separately under identical conditions as in the actual test.

The concentrations of the test item were analytically measured in all test medium samples
from both sampling times (0 and 72 hours). From the control samples, one of the duplicate
samples was analyzed from each of the sampling times. All the samples were analyzed
immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:
In order to assess the toxicity of the test substance to algae, water accommodated fractions (WAFs) with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested. A control was tested in parallel. The selection of the loading rates for the test was based on the results of a range-finding test (non-GLP).

Prior to the start of the test, individual dispersions with loading rates of 10, 32 and 100 mg/L were prepared by mixing 11.3, 33.0, 108.4 mg of the test item, respectively, into 100 mL of test water. The dispersion with the loading rate of 3.2 mg/L was prepared by mixing 10.0 mg of the test item into 300 mL of test water. No auxiliary solvent or emulsifier was used. The dispersions were stirred on magnetic stirrers at room temperature in the dark for 3 hours to dissolve a maximal amount of the test item in the test water.

The stirring period of 3 hours was chosen based on the results of a pre-test (non-GLP) in which the maximum concentration of dissolved test item was reached after the stirring period of 3 hours.

After the stirring period of 3 hours, the saturated dispersions were pre-filtered through glass microfiber filters and filtered through membrane filters just before the start of the test. The filtrates were tested on algae as WAFs. Due to technical reasons, the WAF with the lowest two loading rates of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.

The test media were prepared just before addition of the algae. The WAFs were characterized by GC analysis and the actual test item concentrations were measured.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
-Common name: Scenedesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Liquid cultures of Scenedesmus subspicatus were obtained from the Collection of Algal Cultures (SAD, Institute of Plant Physiology, University of Göttingen, Germany.

ACCLIMATION
- Acclimation period: The test was started (0 hours) by inoculation of 5,000 algal cells per mL of test medium. The algal cells were taken from exponentially growing pre-culture, which was set up 3 days prior to the test under the same conditions as in the test.

The flasks were covered with glass dishes and incubated in a temperature controlled water bath at a temperature of 22-23°C, and continuously illuminated at a measured light intensity of 6200 Lux (range: 5800 to 6600 Lux).

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22 – 23°C

pH:

pH 8.3 – 8.4 at start of test, pH 8.69 - 8.9 at end of test.

Nominal and measured concentrations:

Definitive Test: Nominal 0.32, 1.0, 3.2, 10, 32, and 100 mg/L

Details on test conditions:

- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Counter® electronic particle counter.
- Examination of algal cells: after 72 hours of exposure, a sample was taken from the control and from a concentration with reduced algal growth (loading rate of 100 mg/L). The shape and size of the algal cells were examined microscopically in these samples.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

TEST SOLUTIONS

Observations on the test media were carried out during the mixing and testing of the WAFs. No remarkable observations were made concerning the appearance of the test media
TEST ORGANISM
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

- The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Scenedesmus subspicatus after the test period of 72 hours at the highest loading rate of 100 mg/L. Thus, this loading rate was determined as the 72-hour Lowest Observed Effect Concentration (LOEC). The 72-hour No Observed Effect Concentration (NOEC ) was determined to be at the loading rate of 32 mg/L, since up to and including this loading rate the growth rate and yield of the algae was not statistically significantly lower than the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Validity criteria fulfilled:

yes

Conclusions:

In an OECD 201 study on the effect of the test substance on the growth of Scenedesmus subspicatus the EC50 value was determined to be greater than the 100 mg/L loading rate WAF. The LOEC was 100 mg/L and the NOEC was 32 mg/L.

Executive summary:

The influence of the test substance on the growth of the green algal species
Scenedesmus subspicatus was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3 (1992) and the OECD Guideline 201 (2006).

In order to assess the test substance to Scenedesmus subspicatus, water
accommodated fractions (WAFs) with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control (test water without test item) was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 3 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Thereafter, the dispersions were filtered through membrane filters (0.45 µm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAFs with the lowest loading rates of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.

At the start of the test, the measured test item concentrations (based on a main component of the test item) in the test media with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.11, 0.30, 1.1, 5.1, 15 and 33 mg/L, respectively. Until the end of the test, the measured concentrations decreased to values below the limit of quantification of the analytical method of 0.09 mg/L at all loading rates.

Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Scenedesmus subspicatus after the test period of 72 hours only at the highest loading rate of 100 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) was determined to be at the loading rate of 32 mg/L, since up to and including this loading rate the growth rate and yield of the algae were not statistically significantly lower than in the control.

Description of key information

In an OECD 201 study on the effect of the test substance on the growth of Scenedesmus subspicatus the EC50 value was determined to be greater than the 100 mg/L loading rate WAF. The LOEC was 100 mg/L and the NOEC was 32 mg/L. 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L