Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Remarks:

Scenedesmus subspicatus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2000 to 01 December 2000

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: OP 1/86, Vers. 10/86
- Expiration date of the lot/batch: August 22, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (at RCC: at about 20 °C, away from direct sunlight)
- Solubility of the test substance in water: >50 g/l at 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.

Vehicle:

no

Details on test solutions:

The test medium of the highest test item concentration of nominal 100 mg/L was prepared by dissolving 60 mg of the test item completely in 600 mL test water by stirring for 15 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.

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 "Sammlung von Algenkulturen" (Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-lnstitut für Pflanzenwissenschaften, Universität 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:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 °C

pH:

7.9

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
Analytical concentrations: varying in the range from 96 to 101 % of nominal.

Details on test conditions:

Experimental part A:
The algae (1E+04 cells/mL) 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 due to 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 due to 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.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

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 (growth rate u and biomass b) 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 u and the and mean biomass b of the algae were 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 32 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.
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 very similar inhibition effect on the algal growth was observed compared to experimental part A The algal growth was statistically significantly reduced compared to the control after 72 hours test period first at the test concentration of 32 mg/L.

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.

The differences between the results of experimental parts A and B were described for each test concentration as percentage inhibition of the growth rate µA (IµA) minus the percentage inhibition of the growth rate µB (IµB) 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 µA/µB 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 %, respectively the quotient µA/µB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates µA and µB are essentially the same.

At all test concentrations of this test the differences of the growth rates µA and µB are lower than 10 % and the quotients µA/µB are at least 0.9.

Validity criteria fulfilled:

yes

Conclusions:

This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40224/C on Scenedesmus subspicatus was caused only by the indirect effect, the light absorption in the colored test solutions. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus can be excluded up to the highest test concentration of 100 mg/L.

Executive summary:

The influence of the test substance on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the 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 and 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 96 to 101 % of the nominal values. The test substance was stable in the test media under the test conditions during the test period of 72 h. Therefore, all biological results are related to the nominal concentrations 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 substance 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 influence of the test substance on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the 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 and 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 96 to 101 % of the nominal values. The test substance was stable in the test media under the test conditions during the test period of 72 h. Therefore, all biological results are related to the nominal concentrations 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 substance 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.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L