trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

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:

28 July 2006 to 21 September 2006

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 to quantify not only 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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

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); - duplicate samples from the control (without algae). After 72 hours: - duplicate samples from each test medium (without algae); - 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 duplicate test media samples form the nominal test concentrations of 10-100 mg/L from both sampling times (0 and 72 hours). The samples from the test concentration of 4.6 mg/L were not analyzed, since this concentration was below the determined 72-hour NOEC. 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 nominal 100 mg/L was prepared by dissolving 60.4 mg of the test item completely in 600 mL of test water using 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.

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:

24 mg/L as CaC03.

Test temperature:

23 °C

pH:

7.9-8.9

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal 4.6, 10, 22, 46 and 100 mg/L, analysed test media data varied in the range from 83 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 three 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 solution. All flasks were incubated in a temperature controlled water bath at 23 °C and continuously illuminated at a measured light intensity of about 8500 Lux, range: 7070 to 9250 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.

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. For the quantification of the algicidal effects versus the growth inhibition due to reduced light intensities, the percentage inhibition of algal biomass and growth rate (compared to the control) was calculated for each test concentration. The growth rate was compared between both experimental parts A and B.

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 100mg/L). The shape of the algal cells was examined microscopically in these samples.

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:

NOEC

Effect conc.:

10 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:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Experimental part A:
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 22 mg/L (results of a Dunnett-test, one-sided, a = 0.05, Table 5). 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 (data not shown). The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 100 mg/L) in experimental part A and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.
Experimental part B:
In experimental part B a very similar inhibition effect on the algal growth was observed compared to experimental part A.

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 the percentage inhibition of the growth rate rA (IrA) minus the percentage inhibition of the growth rate rB (IrB) after the 72 hours test period. At all test concentrations this difference was 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 difference between inhibition in experimental part A and B should not be 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 difference between the inhibition of growth rates rA and rB is lower than 10 % and the quotient rA/rB is 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 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 Scenedesmus subspicatus can be excluded up to the highest test concentration of 100 mg/L.

Executive summary:

In a GLP-compliant study, the influence of FAT 40826/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU method C.3 and the OECD Guideline 201. 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 4.6, 10, 22, 46 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were colored by the test item. The analytically determined test item concentrations in the analyzed test media varied in the range from 83 to 100 % of the nominal values. Consequently, the test item was stable 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. 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 72-h ErC50 of test substance is 100 mg/L while NOEC is 10 mg/L in freshwater algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

In a GLP-compliant study, the influence of FAT 40826/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU method C.3 and the OECD Guideline 201. 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 4.6, 10, 22, 46 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were colored by the test item. The analytically determined test item concentrations in the analyzed test media varied in the range from 83 to 100 % of the nominal values. Consequently, the test item was stable 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. 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.