Tetrasodium 5-[4-chloro-6-(N-ethyl-anilino)-1,3,5-triazin-2-ylamino]-4-hydroxy-3-(1,5-disulfonatonaphthalen-2-ylazo)-naphthalene-2,7-disulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Remarks:

Toxicity to Scenedesmus subspicatus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 08, 1994 to Nov 10, 1994

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 material and methods incl. tables"

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See "Any other information on material and methods incl. tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
SR 6947/14
- Expiration date of the lot/batch:
July 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
at room temperature.
- Stability under test conditions:
in water: according to RCC Project 205648 stable over at least 96 hours.
- Solubility of the test substance in the solvent/vehicle:
>300 g/l in water.

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test substance concentrations duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control. All samples are kept stored at -20 °C to enable additional analyses on request of the sponsor.

Vehicle:

no

Details on test solutions:

The test medium of the highest test concentration was prepared just before the start of the test by dissolving the test substance in test water (100 mg/L). Adequate volumes of this intensively mixed test medium were added to test water to prepare the following nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance). The test concentrations were based on the results of a range-finding 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, Pflanzenphysiologisches Institut der Universitiit Gottingen, D-37073 Gottingen, F.R.G. The algae were grown in the laboratories of RCC 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 - 24.1 °C

pH:

7.9 - 10.4

Nominal and measured concentrations:

Nominal concentration: 1.0, 3.2, 10, 32, 100 mg/L

Details on test conditions:

The test was performed in Erlenmeyer flasks (50 ml), each with 50 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:
- Experimental part A: The algae grew in test media with dissolved dyestuff in the Erlenmeyer flasks (The test medium of the highest test substance concentration was prepared by dissolving the test substance in test water (100 mg/L). Adequate volumes of this intensively mixed test medium were added to test water to prepare the test media of the following nominal concentrations: 1, 3.2, 10, 32 and 100 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance). The test media were prepared just before the start of the test). All glass dishes above the cylinders contained untreated test water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the test substance and in addition to the reduced light intensities in the coloured test media in the Erlenmeyer flasks.

- Experimental part B: In this experimental part the glass dishes above the cylinders contained the coloured dyestuff solutions with the same five 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 substance (as in the control), however under changed light conditions due to the filter effect of the coloured 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 20 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. All flasks were incubated in a temperature controlled water baths and continuously illuminated at a mean light intensity of about 8200 Lux, range 7600-8600 Lux. The light intensity was measured just before the start of the test below the coating cylinders at nine places in the area.

All flasks were incubated in a temperature controlled water bath and continuously illuminated at a mean light intensity of 8200 Lux, range 7600 - 8600 Lux. The light intensity was measured just before the start of the test below the coating cylinders at nine places in the area, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (universal white L 25, 36 W) installed above the algal flasks.

Counting and examination of algal cells: Samples of 1 ml test solution were taken out of all flasks after 24, 48 and 72 hours of exposure and not replaced. The algae cell densities in the samples were determined by counting with an electronical particle counter (AL CELLCOUNTER, Model 871, ALSysteme, D-76149 Karlsruhe), three measurements per flask and time. In addition, a sample was taken from the control and from the highest test concentration in experimental part A with reduced algal growth (nominal 100 mg test substance/L) after a test period of 72 hours. The shape of the treated algal cells was microscopically examined and compared with the cells in the control.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- In experimental part A, the 72-h LOEC (lowest concentration tested with a statistically significant* inhibition effect after the 72 h test period) for both growth parameters the algal biomass b and the growth rate µ amounted to 3. 2 mg test substance/L, the 72-h NOEC (highest concentration tested without a statistically significant inhibition effect) on these parameters amounted to 1.0 mg/L.

- In experimental part B (pure light filter effect) the 72-h LOEC for the algal biomass b was 3.2 rng test substance/L, the 72- h NOEC was 1.0 mg/L. The growth rates µ in this experimental part were slightly less affected, the 72-h LOEC for this parameter amounted to 10 mg/L, the 72-h NOEC to 3.2 mg/L. For the quantification of the algicidal effect versus the growth inhibition due to the pure light filter effect, the differences between the percentages of the inhibition rates in experimental parts A and B after the 72 h test period were calculated. For the algal biomass these differences varied in the range of 1.8 % to 42.5 %, for the growth rate µ between 0.5 % and 23 %. These moderate differences between the inhibition rates in parts A and B were obviously caused by a real toxic effect of the test substance on the algal growth. Thus, after correction of the total inhibition effect by the light filter effect of the dyestuff, the real toxic effect of FAT 40'406/A on the growth of Scenedesmus subspicatus after the 72h exposure period amounted to in maximum about 43% up to the highest test concentration of 100 mg test substance/L. The rest of the of the total inhibition effect, determined in experimental part A (as in a usual algal growth inhibition test) was caused by the pure light filter effect of the dyestuff. At the microscopical examination of the shape of the algal cells after 72 h incubation period no difference was observed between the algae in the test concentration of nominal 100 mg test substance/L in experimental part A and the algal cells in the control. Thus, a modification of the shape of the algal cells, growing in the test solutions with this high test substance concentration could not be observed. In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance FAT 40'406/A on Scenedesmus subspicatus was caused in a high degree due to the indirect effect, the light absorption in the coloured test solutions. The real toxic effect of the test substance amounted to in maximum about 43 % growth inhibition up to the highest test concentration of 100 mg/L. Thus, the 72-hour EuC50 and EbC50 for FAT 40'406/A could be determined to be higher than 100 mg test substance/L when based on the real toxic effect. In the control the cell density has increased from nominal N = 1.0E+4 cells/ml at the start of the test (0 hours) to N = 78.93E+4 cells/ml (mean value) after 72 hours by a factor of approximately 79. Thus, the algal growth in the control was sufficiently high. At the start of the test, the pH-value in the test media was always pH 7.9, at the end of the test pH-values were measured between pH 8.5 and 10.4. This increase of the pH-values was obviously caused by the CO2-consumption of algae during their rapid growth respectively their high densities (although the test flasks have been intensively stirred). The analytically determined test substance concentrations in the test media of all test concentrations varied in the range from 99.5 % to 104.1 % of the nominal test concentrations. In the test media the test substance was sufficiently stable at least over the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test substance.

Reported statistics and error estimates:

The EbC50 and EµC50 (the concentrations of the test substance corresponding to 50 % inhibition of algal biomass respectively growth rate compared to the control) and as far as possible the corresponding EC10- and EC90-values and their 95%-confidence limits were calculated by PROBIT ANALYSIS For the determination of the LOEC and NOEC, the calculated mean biomass of algae and mean growth rates "µ" at the test concentrations were tested on significant differences to the control values by Dunnett-tests.

A moderately higher inhibition effect on the algal growth was observed in experimental part A, where the algae grew in the test solutions with dissolved test substance compared to the inhibition effect by the pure light filter effect in experimental part B. Hence, the EC-values based on the results in experimental part B were higher than the corresponding EC-values in experimental part A.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EµC50 and EbC50 of FAT 40406/A was determined to be greater than 100 mg/L basis of growth rate and biomass.

Executive summary:

The influence of the test substance on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-h static test according to the OECD Guideline No. 201 adopted in June 7, 1984, and the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992. The test was performed in compliance with GLP. The test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algal cells and due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions. Thus two experimental parts were developed. 

Experimental part A - where the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the dyestuff and in addition to the reduced light.

Experimental part B - where inhibition in part B was caused due to the pure light filter effect intensities in the coloured test media in the Erlenmeyer flasks.

The nominal test concentrations were 0 (control), 1, 3.2, 10, 32 and 100 mg test substance/L. All test media down to the lowest test concentration were little to strongly coloured by the test substance. A moderately higher inhibition effect on the algal growth was observed in experimental part A, where the algae grew in the test solutions with dissolved test substance compared to the inhibition effect by the pure light filter effect in experimental part B. Hence, the EC-values based on the results in experimental part B were higher than the corresponding EC-values in experimental part A:

 

Biomass in:

Experimental part A:

- E_bC10 (0 - 72 h): 0.8 mg/L (95 % CF: 0.1 - 8.6 mg/L)

- E_bC50 (0 - 72 h): 6.5 mg/L (95 % CF: 1.8 - 23.2 mg/L)

- E_bC90 (0 - 72 h): 56.6 mg/L (95 % CF: 6.6 - 484.3 mg/L)

Experimental part B

- E_bC10 (0 - 72 h): 3.1 mg/L (95 % CF: 0.5 - 21.1 mg/L)

- E_bC50 (0 - 72 h): 22.5 mg/L (95 % CF: 6.6 - 77.2 mg/L)

- E_bC90 (0 - 72 h): 163.8 mg/L (95 % CF: 15.2 - 1762.9 mg/L)

 

Growth rate µ in:

Experimental part A

- E_µC50 (0 - 72 h): 69.3 mg/L (95 % CF: 5.7 - 836.7 mg/L)

- E_µC10 (0 - 72 h): 3.0 mg/L (95 % CF: 0.2 - 52.7 mg/L)

Experimental part B

- E_µC50 (0 - 72 h): 158.5 mg/L (95 % CF: 8.1 - 3116.9 mg/L)

- E_µC10 (0 - 72 h): 14.2 mg/L (95 % CF: 1.5 - 133.7 mg/L)

 

In experimental part A, the 72-h LOEC (lowest concentration tested with a statistically significant inhibition effect after the 72 h test period) for both growth parameters, the algal biomass b and the growth rate µ, amounted to 3. 2 mg test substance/L, the 72-h NOEC (highest concentration tested without a statistically significant inhibition effect) on these parameters amounted to 1.0 mg/L.

In experimental part B (pure light filter effect) the 72 -h LOEC for the algal biomass b was 3.2 mg test substance/L, the 72 -h NOEC was 1.0 mg/L (as in experimental part A). The growth rates µ in this experimental part were slightly less affected, the 72 -h LOEC for this parameter amounted to 10 mg/L, the 72 - h NOEC to 3.2 mg/L.

For the quantification of the algicidal effect versus the growth inhibition due to the pure light filter effect, the differences between the percentages of the inhibition rates in experimental parts A and B after the 72 h test period were calculated. For the algal biomass these differences varied in the range of 1.8 % to 42.5 %, for the growth rate µ between 0.5 % and 23.0 %. These moderate differences between the inhibition rates in parts A and B were obviously caused by a real toxic effect of the test substance on the algal growth. In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in a high degree due to the indirect effect, the light absorption in the coloured test solutions. The real toxic effect of the test substance amounted to in maximum about 43 % growth inhibition up to the highest test concentration of 100 mg/L. Thus, the 72-h E_µC 50 and E_bC 50 for the test substance could be determined to be higher than 100 mg test substance/L when based on the real toxic effect. The analytically determined test substance concentrations in the test media of all test concentrations varied in the range from 99.5 % to 104.1 % of the nominal test concentrations. In the test media the test substance was sufficiently stable at least over the test period of 72 h. Therefore, all biological results are related to the nominal concentrations of the test substance.

Description of key information

The 72-h ErC50 and EuC50 is >100 mg/L respectively in freshwater algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

14.2 mg/L

Additional information

In a key study the light interference was adjusted was performed to further assess the toxicity of FAT 40406 to algae. The influence of the test substance on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-h static test according to the OECD 201 and EU C.3 guideline. The test was performed in compliance with GLP. The test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algal cells and due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions. Thus two experimental parts were developed; Experimental part A - where the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the dyestuff and in addition to the reduced light. Experimental part B - where inhibition in part B was caused due to the pure light filter effect intensities in the coloured test media in the Erlenmeyer flasks. The nominal test concentrations were 0 (control),1, 3.2, 10, 32 and 100 mg test substance/L. All test media down to the lowest test concentration were little to strongly coloured by the test substance. A moderately higher inhibition effect on the algal growth was observed in experimental part A, where the algae grew in the test solutions with dissolved test substance compared to the inhibition effect by the pure light filter effect in experimental part B. Hence, the EC-values based on the results in experimental part B were higher than the corresponding EC-values in experimental part A:

 

Biomass in:

Experimental part A:

- E_bC 10 (0 - 72 h): 0.8 mg/L (95 % CF: 0.1 - 8.6 mg/L)

- E_bC 50 (0 - 72 h): 6.5 mg/L (95 % CF: 1.8 - 23.2 mg/L)

- E_bC 90 (0 - 72 h): 56.6 mg/L (95 % CF: 6.6 - 484.3 mg/L)

Experimental part B

- E_bC 10 (0 - 72 h): 3.1 mg/L (95 % CF: 0.5 - 21.1 mg/L)

- E_bC 50 (0 - 72 h): 22.5 mg/L (95 % CF: 6.6 - 77.2 mg/L)

- E_bC 90 (0 - 72 h): 163.8 mg/L (95 % CF: 15.2 - 1762.9 mg/L)

 

Growth rate µ in:

Experimental part A

- E_µC 50 (0 - 72 h): 69.3 mg/L (95 % CF: 5.7 - 836.7 mg/L)

- E_µC 10 (0 - 72 h): 3.0 mg/L (95 % CF: 0.2 - 52.7 mg/L)

Experimental part B

- E_µC 50 (0 - 72 h): 158.5 mg/L (95 % CF: 8.1 - 3116.9 mg/L)

- E_µC 10 (0 - 72 h): 14.2 mg/L (95 % CF: 1.5 - 133.7 mg/L)

 

In experimental part A, the 72-h LOEC (lowest concentration tested with a statistically significant inhibition effect after the 72 h test period) for both growth parameters, the algal biomass b and the growth rateµ,amounted to 3. 2 mg test substance/L, the 72-h NOEC (highest concentration tested without a statistically significant inhibition effect) on these parameters amounted to 1.0 mg/L. In experimental part B (pure light filter effect) the 72 -h LOEC for the algal biomass b was 3.2 mg test substance/L, the 72 h NOEC was 1.0 mg/L (as in experimental part A). The growth ratesµin this experimental part were slightly less affected, the 72 h LOEC for this parameter amounted to 10 mg/L, the 72 h NOEC to 3.2 mg/L. For the quantification of the algicidal effect versus the growth inhibition due to the pure light filter effect, the differences between the percentages of the inhibition rates in experimental parts A and B after the 72 h test period were calculated. For the algal biomass these differences varied in the range of 1.8 % to 42.5 %, for the growth rate µ between 0.5 % and 23.0 %. These moderate differences between the inhibition rates in parts A and B were obviously caused by a real toxic effect of the test substance on the algal growth. In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused in a high degree due to the indirect effect, the light absorption in the coloured test solutions. The real toxic effect of the test substance amounted to in maximum about 43 % growth inhibition up to the highest test concentration of 100 mg/L. Thus, the 72-h E_µC₅₀ and E_bC₅₀ for the test substance could be determined to be higher than 100 mg test substance/L when based on the real toxic effect. The analytically determined test substance concentrations in the test media of all test concentrations varied in the range from 99.5 % to 104.1 % of the nominal test concentrations. In the test media the test substance was sufficiently stable at least over the test period of 72 h. Therefore, all biological results are related to the nominal concentrations of the test substance. 

In another supporting study, the influence of FAT 40406/A on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 96-h static test according to the OECD Guideline No. 201 adopted June 7, 1984. The test was performed in compliance with GLP. The nominal test concentrations were 0, 10, 32, 100, 320 and 1000 mg test substance/L. FAT 40406/A had a inhibition effect on the growth of Scenedesmus subspicatus. The inhibition was calculated for both growth parameter, the algae biomass (determined as the area under the growth curve) and the growth rateµ.Since in the control no further exponential growth was observed after 72 to 96 h, only the results after 72 h test duration were reported:

 

Biomass:

- EbC 50 (0 - 72 h): 27.6 mg test substance/L (95 % CF: 9.9 - 63.4 mg test substance/L)

- EbC 10 (0 - 72 h): 1.4 mg test substance/L (95 % CF: 0.1 - 4.8 mg test substance/L)

- EbC 90 (0 - 72 h): 552.7 mg test substance/L (95 % CF: 195 - 4864 mg test substance/L)

Growth rate µ:

- EµC 50 (0 - 72 h): 1193 mg test substance/L (9 5% CF: 647 -2201 mg test substance/L)

- EµC 10 (0 - 72 h): 23.8 mg test substance/L (95 % CF: 13.7 -41.2 mg test substance/L)

- EµC 90 (0 - 72 h): >1000 mg test substance/L

 

The test substance concentrations in the analysed test media amounted to 95.1 - 106.9 % of the nominal values. In the test media, incubated under test conditions (but without algae), the test substance was sufficiently stable. Therefore, the reported results are related to the nominal concentration of the test substance.