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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:

Experiment start date - 20 April 2003; Experiment completion date - 16 May 2003; Study completion date - 07 July 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:

The test method was modified following a method developed by RCC Ltd (Ref. 1) to quantify the algicidal effect of colored test substances, but also the growth inhibition effect caused by reduced light intensities in the colored test media.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The test method was modified following a method developed by RCC Ltd (Ref. 1) to quantify the algicidal effect of colored test substances, but also the growth inhibition effect caused by reduced light intensities in the colored test media.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 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 test start 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.

Vehicle:

no

Details on test solutions:

The test item (29.91 mg) was dissolved in purified water and made up to the mark in a 100 ml volumetric flask to prepare a stock solution of 299 mg/L. Defined volumes of this stock solution were diluted with a mixture of acetonitrile/purified water (70:30; v:v) to obtain standard solutions in the range of 0.897 to 29.9 mg/l of the test item.

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 Gottingen, 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:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

23 °C throughout the test period

pH:

8.0 - 8.2 at beginning and 8.2 - 9.6 at the end of test

Nominal and measured concentrations:

Nominal concentrations : 0 (control), 1.0, 3.2, 10, 32, 100 mg/L
Measured values were in the range of 93 - 110 % of nominal values throughout the test period and thus test item was considered stable during performance of the test.

Details on test conditions:

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). All 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.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 46 mg/L

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

growth rate

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Dunnett-test, one-sided, α = 0.05

Analytical results:

The analytically determined test item concentrations in the analyzed test media varied in the range from 93 to 110 % of the nominal values at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item.

 

Experimental part A: 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 32 mg/L (results of a Dunnett-test, one-sided, a= 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 10 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. For the biomass b, the same LOEC- and NOEC-values were determined. The EC values were calculated for the algal biomass b and the growth rate r after 72 hours test duration:

 

Experimental part A

 

Parameter (0-72 h)	Blomass b (mg/l)	Growth rate r (mg/l)
EC50	46	>100
EC10	4.8	20
NOEC	10	10
LOEC	32	32

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 100 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.

 

Experimental part B: In experimental part B the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified. In this experimental part a nearly identical inhibition effect on the algal growth was observed compared to experimental part A. The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A. The EC values in experimental part 8 are listed below:

 

Parameter (0-72 h)	Biomass b (mg/L)	Growth rate r (mg/L)
EC50	44	>100
EC10	2.8	17

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 8 was based on the growth rates. The differences between the results of experimental parts A and B were described for each test concentration as percentage inhibition of the growth rate rA (lrA) minus the percentage inhibition of the growth rate rB (Ire) after the 72 hours test period. At all test concentrations these differences were lower than 10 %. As another measure of difference, the quotient of the growth rates rA/rB was calculated for each test concentration. At all test concentrations this quotient was at least 0.9 or higher. 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 all test concentrations of this test, the differences of the inhibition of growth rates rA and rB are lower than 10 % and the quotients rA/rB are at least 0.9. 

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (scenedesmus subspicatus, 72h): >100 mg/L

Executive summary:

The influence of the test item FAT 40810/A 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, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The analytically determined test item concentrations in the analyzed test media varied in the range from 93 to 110 % of the nominal values at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item. The same 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 as in the second parallel experimental part, where the algae grew in the test media with dissolved test item. Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40810/A on Scenedesmus subspicatus was caused only by an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 100 mg/L.

Description of key information

The NOEC and ErC50 towards scenedesmus subspicatus was set to >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The influence of the test item FAT 40810/A 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, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The analytically determined test item concentrations in the analyzed test media varied in the range from 93 to 110 % of the nominal values at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item. The same 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 as in the second parallel experimental part, where the algae grew in the test media with dissolved test item. Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40810/A on Scenedesmus subspicatus was caused only by an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 100 mg/L.