Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate

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:

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:

no

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

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.

For the determination of the stability of the test substance under the test conditions, sufficient volumes of all freshly prepared test media and the control were incubated under the same conditions as in the actual test (but without algae) and sampled in duplicate at the end of the test (after 72 hours test period).

The concentrations of the test substance FAT 45168/A were measured in the duplicate test media samples from all test concentrations from both sampling dates (0 and 72 hours). From the control samples only one of the duplicate samples was analysed from each of both sampling dates (0 and 72 hours).

All samples are kept stored at -20 °C to enable additional analyses on request of the sponsor. After delivery of the final test report all samples will be discarded.

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 amounts of this intensively mixed test medium were added to test water to prepare the following test concentrations: 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/l. Additionally, a control (test water without addition of the test substance) was tested in parallel.

The test concentrations were based on the results of a range-finding test. The range-finding test was not performed in compliance with the GLP-Regulations, but the raw data of the rangefinding test will be archived under the RCC Project number of the present study.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: No. 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, D-37073 Göttingen, Germany
- Method of cultivation: 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

Post exposure observation period:

Study terminated post 72 hours exposure

Hardness:

No data

Test temperature:

24 - 24.2 °C

pH:

Study start: 8.
Study end: 10.1

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

1.0 mg/L
3.2 mg/L
10.0 mg/L
32.0 mg/L
100.0 mg/L

Details on test conditions:

The test was started (0 hours) with a biomass of nominal 10.000 cells per ml test solution. These cells were taken from an exponentially growing pre-culture, which was set up about 7 2 hours prior to the test at the same conditions as in the test.

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. 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 dyestuff 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 dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes. Thus, the growth inhibition in part B was caused due to light absorption only. The depth of the test solutions in the glass dishes was 20 mm, i.e. half the depth of the test solutions in the Erlenmeyer flasks because the algae in the stirred test solutions stay in the statistical mean in this mean depth.

All flasks were incubated in a temperature controlled water bath and continuously illuminated at a mean light intensity of 8283 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.

The test duration was 72 hours.

Reference substance (positive control):

no

Key result

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All test media down to the lowest test concentration of nominal 1.0 mg test substance/L were little to strongly coloured by the test substance.
A slightly higher inhibition effect on the algal growth was observed in experimental part A, where the algae grew in the test solu tions with dissolved test substance compared to the inhibition effect only by light absorption in experimental part. Hence, the EC-values based on the results in experimental part B were slightly higher than the corresponding EC-values in experimental part A:

In experimental part A, the 72-hour LOEC (lowest concentration tested with a statistically significant* inhibition effect after the 7 2 hours test period) for both growth parameters, the algal biomass b and the growth rate p. amounted to 3.2 mg test substance/ L, the 72-hour NOEC (highest concentration tested without a statistically* significant inhibition effect) on these parameters amounted to 1.0 mg/L.

In experimental part B (light effect only), the 72-hour LOEC for the growth rate u amounted as in experimental part A to 3.2 mg/L, the 72-hour NOEC to 1.0 mg/L. The growth parameter algal biomass in experimental part B was slightly more affected, here the 72- hour LOEC was determined at 1.0 mg/L, the 72-hour NOEC was lower than 1.0 mg/L.
* results of Dunnett-tests, one-sided, p < 0.05

For the quantification of an 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 hours test period were calculated. For the biomass of algae these differences varied in the range of 1.0 to 32.1 %, for the growth rate "micro" between 0.4 and 17.3 %.

These slight differences between the inhibition rates in parts A and B might be caused by a real toxic effect of the test substance on the algal growth. However, at the highest test concentration of 100 mg/L these differences were rather small (4.2 and 7.9 %, respectively). Thus, after correction of the total inhibition effect by the filter effect of the dyestuff, the real toxic effect of FAT 45168/A on the growth of Scenedesmus subspicatus after the 7 2 hours exposure period amounted to in maximum about 32 % up to the highest test concentration of 100 mg test substance/L. The rest of the total inhibition effect, determined in experimental part A (as in a usual algal growth inhibition test) was clearly caused by the pure light filter effect of the dyestuff.

At the microscopical examination of the shape of the algal cells after 72 hours incubation period no difference was observed between the algae in the test concentration of nominal 32.0 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 solutions with this dissolved 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 45168/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 32 % growth inhibition up to the highest test concentration of 100 mg/L. Thus, the 72-hours EC 50 (Biomass) and EC 50 (Growth rate) for FAT 45168/A could be determined to be clearly higher than 100 mg test substance/L when based on the real toxic effect.

In the control the cell density has increased after 72 hours by a factor of approximately 123. 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 8.0, at the end of the test pH-values were measured between pH 8.1 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 varied in the range from 78.5 to 101.0 % of the nominal concentrations. The mean values of the test substance concentrations, measured in the samples from the start and the end of the test for each test concentration, amounted from 88.9 to 98.8 % of the nominal values. Therefore, all reported biological results are related to the nominal concentrations of the test substance.

Results with reference substance (positive control):

None

Reported statistics and error estimates:

For both experimental parts also the EbC50 and EuC50 (the concentrations of the test substance corresponding to 50 % inhibition of algae biomass respectively growth rate compared to the control), the corresponding EC10- and EC90-values and their 95 %-confidence limits were calculated by Probit Analysis.

For the determination of the LOEC/NOEC, the calculated mean biomass of algae (= areas under the growth curves) and mean growth rates "micro" in the test concentrations were tested on significant differences to the control value by the DUNNETT-TEST.

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

For both experimental parts also the EC50 (Biomass) and EC50 (Growth rate)

the concentrations of the test substance corresponding to 50 % inhibition of algae biomass respectively growth rate compared to the control), the corresponding EC10- and EC90-values and their 95%-confidence limits were calculated by Probit Analysis.

For the determination of the LOEC/NOEC, the calculated mean biomass of algae (= areas under the growth curves) and mean growth rates "micro" in the test concentrations were tested on significant differences to the control value by the Dunnett-Test.

Validity criteria fulfilled:

yes

Conclusions:

72-hours EC 50 (Biomass) and EC 50 (Growth rate) for FAT 45'168/A could be determined to be clearly higher than 100 mg test substance/L when based on the real toxic effect.

Executive summary:

The influence of the test substance FAT 45'168/A on the growth of the green algae Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984, and the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992. However, 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 or due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions. The test was performed in compliance with Good Laboratory Practice Regulations.

The test included two experimental parts:

Experimental part A: The algae grew in test media with dissolved dyestuff in Erlenmeyer flasks, each placed in a black cylinder. The cylinders were covered with glass dishes, containing untreated test water in this experimental part.

Experimental part B: The glass dishes above the cylinders contained the coloured dyestuff solutions with the same five test concentrations as in Part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were little to strongly coloured by the test substance.

A slightly 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 only by light absorption in experimental part B.

Hence, the EC-values based on the results in experimental part B were slightly higher than the corresponding EC-values in experimental

part A.

In experimental part A, the 72-hour LOEC (lowest concentration tested with a statistically significant inhibition effect after the 7 2 hours test period) for both growth parameters, the algal biomass b and the growth rate u amounted to 3.2 mg test substance/ L, the 72-hour NOEC (highest concentration tested without a statistically significant inhibition effect) on these parameters amounted to 1.0 mg/L. In experimental part B the 72-hour LOEC for the growth rate also amounted to 3.2 mg/L, the 72-hour NOEC to 1.0 mg/L. For the algal biomass in experimental part B

the 72-hour LOEC was determined at 1.0 mg/L, the 72-hour NOEC was lower than 1.0 mg/L.

The differences between the percentages of the inhibition rates in experimental parts A and B after the 72 hours test period were in the range of 1.0 % to 32.1 % for the biomass of algae, and between 0.4 % and 17.3 % for the growth rate. These slight differences between the inhibition rates in parts A and B might be 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 FAT 45'168/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 32 % growth inhibition up to the highest test concentration of 100 mg/L.

Thus, the 72-hours EC 50 (Biomass) and EC 50 (Growth rate) for FAT 45'168/A could be determined to be clearly higher than 100 mg test substance/L when based on the real toxic effect.

Description of key information

The 72-hours EC50 (Biomass) and EC50 (Growth rate) for FAT 45168/A could be determined to be clearly higher than 100 mg test substance/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The influence of the test substance FAT 45168/A on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984, and the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992. However, 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 or due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions. The test was performed in compliance with Good Laboratory Practice Regulations.

The test included two experimental parts:

Experimental part A: The algae grew in test media with dissolved dyestuff in Erlenmeyer flasks, each placed in a black cylinder. The cylinders were covered with glass dishes, containing untreated test water in this experimental part.

Experimental part B: The glass dishes above the cylinders contained the coloured dyestuff solutions with the same five test concentrations as in Part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were little to strongly coloured by the test substance.

A slightly 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 only by light absorption in experimental part B.

Hence, the EC-values based on the results in experimental part B were slightly higher than the corresponding EC-values in experimental

part A.

In experimental part A, the 72-hour LOEC (lowest concentration tested with a statistically significant inhibition effect after the 72 hours test period) for both growth parameters, the algal biomass b and the growth rate u amounted to 3.2 mg test substance/ L, the 72-hour NOEC (highest concentration tested without a statistically significant inhibition effect) on these parameters amounted to 1.0 mg/L. In experimental part B the 72-hour LOEC for the growth rate also amounted to 3.2 mg/L, the 72-hour NOEC to 1.0 mg/L. For the algal biomass in experimental part B the 72-hour LOEC was determined at 1.0 mg/L, the 72-hour NOEC was lower than 1.0 mg/L.

The differences between the percentages of the inhibition rates in experimental parts A and B after the 72 hours test period were in the range of 1.0 % to 32.1 % for the biomass of algae, and between 0.4 % and 17.3 % for the growth rate. These slight differences between the inhibition rates in parts A and B might be 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 FAT 45168/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 32 % growth inhibition up to the highest test concentration of 100 mg/L.

Thus, the 72-hours EC 50 (Biomass) and EC 50 (Growth rate) for FAT 45'168/A could be determined to be clearly higher than 100 mg test substance/L when based on the real toxic effect.