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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
Study period:
Experiment start date - 20 June 2003; Experiment completion date - 26 November 2003; Study completion date - 10 December 2003.
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 40812/A
Batch: WP 8/03
Purity: approx 75%
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C
Analytical monitoring:
yes
Details on sampling:
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. Based on pre-experiments for investigation of the storage stability (without GLP) the test item is sufficiently stable in the test water under these storage conditions. The concentrations of the test item FAT 40812/A were measured in the duplicate test media samples from all test concentrations. 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 concentration of 100 mg/L was prepared by dissolving 90.4 mg of the test item completely in 900 mL of test water using stirring for 10 minutes at room temperature. In a series of subsequent dilution steps this highest test medium was diluted with test water to prepare the test media with the lower test concentrations as stated above. 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 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
- Measured: Analysed test media data varied in the range from 83 to 116% of the nominal values. The mean measured concentrations (calculated as the average over all measurements per test concentration) ranged from 94 to 115% of nominal.
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: 8220 to 9470 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 (six 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 because the algae in the stirred test media stay in the statistical mean in this mean depth.

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 10 mg/L). The shape of the algal cells was examined microscopically in these samples. The test concentration of nominal 10 mg/L was chosen since at the higher concentrations of nominal 32 and 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.:
> 100 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.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Experiment B
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.2 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: 2.1-7.0 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.5 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: 3.4-8.1 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:
growth rate
Remarks on result:
other: Experiment A
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Experiment A, 95% CL: 9.7-36 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Experiment B, 95% CL: 20-36 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.76 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Experiment A, 95% CL: 0.19-1.7 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Experiment B, 95% CL: 0.71-1.6 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 corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test item and/or by the reduced light intensities due to the light absorption in the colored test media.
In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate "r" of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.2 mg/L (results of a Dunnett-test, one-sided, alpha = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the concentration of 1.0 mg/L, since up to and including this test concentration the mean growth rate "r" of the algae was statistically not significantly lower than in the control. 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 10 mg/L in experimental part A and the algal cells in the control. Thus, the shape and size of the algal cells, growing at this concentration of dissolved test item were obviously not affected.
- The EC values in experimental B 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.

Comparison between the results in experimental parts A and B:

According to the recommendations of the Ad-hoc working group of experts on algal growth inhibition for the interpretation of test results of colored substances the comparison between the results in experimental parts A and B was based on the growth rates.

At the lower test concentrations these differences were lower than 10%. However, at the highest test concentration of 100 mg/L the difference was higher than 10%.

As another measure of difference, the quotient of the growth rates rA/rB was calculated for each test concentration. This quotient was also high (higher than 0.9) at the test concentrations from 0.32 to 32 mg/L, but lower than 0.9 at the test concentrations of 100 mg/L.

Differences in growth rates up to the magnitude of 10% are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the recommendations of the Ad-hoc working group of experts on algal growth inhibition tests for colored substances the differences between inhibition in experimental part A and B should be not higher than 10%, and the quotient rA/rB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates rA and rB are essentially the same.

At the highest test concentration of this test, the differences of the inhibition of growth rates rA and rB are higher than 10% and the quotients rA/rB are lower than 0.9. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus can not be excluded at these test concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The 72-h ErC50 is >100 mg/L and the 72-h ErC10 is 4.2 mg/L 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, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of the test substance. The analytically determined test substance concentrations varied from 83% to 116% of nominal values. The mean measured concentrations (calculated as the average over all measurements per test concentration) ranged from 94 to 115% of nominal. Consequently, the test item concentrations were sufficiently constant during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentration of the test item.

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 growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the colored test solutions. However, at the highest test concentration a real toxic effect of the test item on the growth of Scenedesmus subspicatus cannot be excluded.

Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test item on the growth of Scenedesmus subspicatus. Therefore, the results of experimental part A, where the algae grew in the test media with dissolved test item 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 17 mg/L respectively. The ErC10 and EbC10 values were 4.2 mg/L and 0.76 mg/L respectively and the NOEC was 1 mg/L for both endpoints.

Description of key information

The 72-h ErC50 is >100 mg/L and the 72-h ErC10 is 4.2 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:
4.2 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, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of the test substance. The analytically determined test substance concentrations varied from 83 % to 116 % of nominal values. The mean measured concentrations (calculated as the average over all measurements per test concentration) ranged from 94 to 115 % of nominal. Consequently, the test item concentrations were sufficiently constant during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentration of the test item. 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 growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the colored test solutions. However, at the highest test concentration a real toxic effect of the test item on the growth of Scenedesmus subspicatus cannot be excluded. Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test item on the growth of Scenedesmus subspicatus. Therefore, the results of experimental part A, where the algae grew in the test media with dissolved test item 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 17 mg/L respectively. The ErC10 and EbC10 values were 4.2 mg/L and 0.76 mg/L, respectively and the NOEC was 1 mg/L for both endpoints.

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