2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

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:

08 June 2001 to 22 June 2001

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)

Principles of method if other than guideline:

The test method was modified following a method developed by RCC Ltd 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)

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.

Vehicle:

no

Details on test solutions:

The test medium of the highest test item concentration of nominal 100 mg/L was prepared by dissolving 50 mg of the test item completely in 500 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 (=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 "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 - 8.3

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L.
Analysed test media data varied in the range from 96 to 100 % of the nominal values.

Details on test conditions:

Experimental conditions:
The test was started (0 hours) by inoculation of 10,000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test under the same conditions as in the test. 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. To reduce the loss of water and to avoid the entry of dust into the solutions, on top of each cylinder glass dishes, covered with watch glass dishes, were placed. 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 (mean value), range: 8200 to 9200 Lux (minimum and maximum value of measurements at 9 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 vessels were labeled with the RCC study number and all necessary additional information to assure unmistakable identification.

Experimental part A:
The algae grew in test media with dissolved test item in the Erlenmeyer flasks (five test concentrations and a control, see Section 2.5.2). 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.

Counting and Examination of the Algal Cells:
Small volumes of the test media and the control (1.0-2.0 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 100 mg/L). The shape of the algal cells was microscopically examined.

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:

biomass

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.:

100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: toxic effect

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 µ of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 10 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 3.2 mg/L, since up to and including this test concentration the mean growth rate µ 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 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. 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 3.2 mg/L. The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A.

Reported statistics and error estimates:

The EbC50 and EµC50 values (the concentrations of the test item corresponding to 50 % inhibition of algal biomass (b) and growth rate (µ) compared to the control, respectively), and the corresponding EC10 value and their 95 %-confidence limits were calculated as far as possible for both experimental parts by Probit Analysis. For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences compared to the control values by a Dunnett-test.

All test media were slightly to strongly colored by the test item. In the control the cell density increased from nominal N = 1 x 10⁴ cells/ml at the start of the test (0 hours) to N = 39 x 10⁴ cells/ml (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase by at least a factor of 16 was fulfilled. At the start of the test, the pH value in the test media and the control was 7.9. At the end of the test, pH values between 8.1 and 8.3 were measured.

Validity criteria fulfilled:

yes

Conclusions:

This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40800/A 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. The EC50 and NOEC of test substance is >100 and 100 mg/L, respectively.

Executive summary:

The influence of the test substance on the growth of the green alga Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the OECD Guideline No. 201and EU method C.3 under GLP compliance. However, the test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect due to reduced light intensities in the coloured test media. The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance. The analytically determined test substance concentrations in the analysed test media varied in the range from 96 % to 100 % of the nominal values. The test substance was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test substance. The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test substance, but under reduced light intensities by the filter effect of the coloured test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test substance. 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 due to an indirect effect, the light filter effect in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to the highest test concentration of 100 mg test substance/L. Based on the test results, the EC50 and NOEC of test substance is >100 and 100 mg/L respectively.

Description of key information

The 72-h ErC50 and NOErC values in the freshwater algae Scenedesmus subspicatus are >100 mg/L and 100 mg/L, respectively.	

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 toxicity of the test substance towards aquatic algae was investigated in a GLP-compliant test according to OECD 201 (RCC, 2001). In this study, freshwater alga (Scenedesmus subspicatus) were exposed for 72 hours to nominal concentrations of 0, 1.0, 3.2, 10, 32 and 100 mg test substance/L under static conditions. In addition, as all test media were slightly to strongly coloured depending on the test substance concentration, alga were exposed to medium without test substance under varying reduced light conditions. This was done to differentiate between a direct toxic effect on algal growth, as induced by the test substance, and an indirect effect on growth caused by light absorption of the coloured test substance in solution. The effects on algal growth were similar in both experimental set-ups which demonstrates that the observed effect on algal growth was attributable only to light absorption caused by colouring of the test solutions. Based on these findings, the 72-h EbC50 and ErC50 values are determined at >100 mg/L. The NOEC is 100 mg/L.