[No public or meaningful name is available]

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:

Experiment start date - 11 February 2005; Experiment completion date - 06 March 2005; Study completion date - 14 June 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(see "any other information on materials and methods")

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(see "any other information on materials and methods")

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Analytical monitoring:

yes

Details on sampling:

For the analysis 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) and duplicate samples from the control (without algae); - after 72 hours: duplicate samples from each test medium (without algae) and duplicate samples from the control (without algae). For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae. For the analysis of the actual test item concentrations duplicate samples without algae were taken from each test concentration and the control just before the start of the test and after 72 hours (end of the test). For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
All samples were deep-frozen (at about -20 °C) immediately after sampling. The concentrations of the test item were measured in the duplicate test media samples from all test concentrations from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analyzed from each of the sampling times (0 and 72 hours).

Vehicle:

no

Details on test solutions:

The test medium of the highest test item nominal concentration of 100 mg/L was prepared by dissolving 79.64 mg of the test item completely in 796.4 mL of test water using ultrasonic treatment (10 minutes) and stirring (10 minutes at room temperature). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before addition of algae (= start of the test).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus chodat Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3.

Test temperature:

24 °C

pH:

7.9-9.4

Nominal and measured concentrations:

nominal 1.0, 3.2, 10, 32 and 100 mg/L, analysed test media data varied in the range from 80 to 100 % of the nominal values.

Details on test conditions:

The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth. The test was performed in Erlenmeyer flasks (50 mL), each filled with 15 mL algal suspension. The flasks were continuously stirred by magnetic stirrers. Per test concentration, 3 flasks were prepared. For the control, 6 flasks were prepared. Each flask was placed in a black cylinder, coated inside with aluminum foil. On the top of each cylinder, glass dishes covered with watch glass dishes were placed to reduce the loss of water and to avoid the entry of dust into the solutions. All flasks were incubated in a temperature controlled water bath at 24 °C and continuously illuminated at a measured light intensity of about 8800 Lux (mean value), range: 8330 to 9600 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area). The light intensity was measured just before the start of the test below the coating cylinders. The illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the algal flasks.

The test included two experimental parts:
- Experimental part A: The algae grew in test media with dissolved test item in the Erlenmeyer flasks (five test concentrations and a control). The glass dishes above the cylinders contained purified water. Thus, the inhibition of algal growth in this experimental part was caused by a real toxic effect of the test item and in addition to the reduced light intensities in the colored test media in the Erlenmeyer flasks.
- Experimental part B: In this experimental part the glass dishes above the cylinders contained the colored test media with the same test concentrations as in part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in test water without test item (as in the control), however under changed light conditions due to the filter effect of the colored test media in the glass dishes. Thus, the growth inhibition in part B was caused by light absorption only. The depth of the test media in the glass dishes was 4 mm, i.e. half the depth of the test media in the Erlenmeyer flasks ( in the statistical mean, the algae stay in this mean depth in the stirred test media).

Counting and Examination of the Algal Cells:
Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample. In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth in experimental part A (nominal 32 mg/L). The shape of the algal cells was examined microscopically in these samples. The test concentration of nominal 32 mg/L was chosen since at the highest concentration of nominal 100 mg/L the algal cell density was too low for a reliable microscopic examination.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

189 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A, 95 % CL: 88 - 1079 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

245 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B, 95 % CL: 138-901 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A, 95 % CL: 4.7-23 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

36 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B, 95 % CL: 21-53 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Experiment A, 95 % CL: 17-87 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Experiment B, 95 % CI: 42-138 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Experiment A, 95 % CL: 0.60-7.7 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Experiment B, 95 % CL: 4.9-26 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Experiment A

Details on results:

- Experimental part A: In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate of Scenedesmus subspicatus after the exposure period of 72 hours at concentrations of 32 mg/L and above (results of a Dunnett-test, one-sided, alpha=0.05). At the microscopical examination of the shape of the algal cells after 72 hours incubation period no difference was observed between the algae growing in the test item concentration of 32 mg/L in experimental part A and the algal cells in the control.
- Experimental part B: In experimental B the EC values were higher than the corresponding EC values in experimental part A. Thus, only a part of the algal growth inhibition was obviously caused by the pure light effect.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 is >100 mg/L and the 72-h ErC10 is 13.0 mg/L, respectively in freshwater alga (S. subspicatus).

Executive summary:

In a GLP-compliant study, performed according to the OECD guideline 201, the influence of the test substance on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-hour static test. The nominal test concentrations were 0, 1.0, 3.2, 10, 32 and 100 mg/L of the test substance. The analytically determined test substance concentrations varied from 80 % to 100 % of nominal values. As all test media were slightly to strongly coloured by the test substance, the test method was slightly modified to determine the growth inhibition effect due to reduced light intensities in the coloured test media. In experiment A, the normal test was performed, but in experiment B, growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media. The result showed that the growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in part due to the indirect effect of light absorption in the colored test solutions. However, the differences between experimental parts A and B were too high to state that the algal growth is inhibited solely as a result of a reduction in light intensity. Therefore, the results of experimental part A were used for the determination of the toxic effect. Based on these findings, the 72-hour ErC50 for growth inhibition and the EbC50 for biomass were determined at >100 mg/L and 32 mg/L respectively. The 72 -h ErC10 and EbC10 values were 13.0 mg/L and 35 mg/L and the 72 -h NOErC and NOEbC were 10 mg/L and 1.0 mg/L, respectively.

Description of key information

The 72-h ErC50 is >100 mg/L and the 72-h ErC10 is 13 mg/L in freshwater alga (S. subspicatus).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

13 mg/L

Additional information

In a GLP-compliant study, performed according to the OECD guideline 201, the influence of the test substance on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-hour static test. The nominal test concentrations were 0, 1.0, 3.2, 10, 32 and 100 mg/L of the test substance. The analytically determined test substance concentrations varied from 80 % to 100 % of nominal values. As all test media were slightly to strongly coloured by the test substance, the test method was slightly modified to determine the growth inhibition effect due to reduced light intensities in the coloured test media. In experiment A, the normal test was performed, but in experiment B, growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media. The result showed that the growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in part due to the indirect effect of light absorption in the colored test solutions. However, the differences between experimental parts A and B were too high to state that the algal growth is inhibited solely as a result of a reduction in light intensity. Therefore, the results of experimental part A were used for the determination of the toxic effect. Based on these findings, the 72-hour ErC50 for growth inhibition and the EbC50 for biomass were determined at >100 mg/L and 32 mg/L respectively. The 72-h ErC10 and EbC10 values were 13.0 mg/L and 3.5 mg/L and the 72-h NOErC and NOEbC were 10 mg/L and 1.0 mg/L, respectively.