Reaction mass of tetrasodium 5-{[4-chloro-6-(4-{[2-(sulfonatooxy)ethyl]sulfonyl}anilino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl) diazenyl]naphthalene-2,7-disulfonate and trisodium 5-({4-chloro-6-[4-(vinylsulfonyl) anilino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October to December 1992

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:

modified to differetiate between toxic effects and indirect effects due to light absorption in coloured test subtrance

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A stock solution was prepared just before the start of the test by dissolving the test substance in test water (1.0 g/l). Adequate amounts of the intensively mixed stock solution were added to test water to prepare the following nominal concentrations: 1.0, 3.2, 10.0, 32.0 and 100 mg/L. Additionally, a control (test water without any additions) was tested. 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:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Method of cultivation: in synthetic test water according to OECD guideline 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

NA

Hardness:

no data

Test temperature:

Start: 23°C
Day 1: 23.3°C
Day 2: 23.6°C
Day 3: 23.8°C

pH:

start: 7.8 to 7.9
end: 8.0 to 10.3

Dissolved oxygen:

no data

Salinity:

NA

Nominal and measured concentrations:

1.0, 3.2, 10.0, 32.0 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed (Erlenmeyer flask covered with glass dishes, covered with wath glass dishes
- Material, size, headspace, fill volume: glass, 100 mL, -, 50 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Number of organisms: 10E04 cells/mL

The test was performed in Erlenmeyer flasks (100 ml), each with 50 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in control Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminum 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 (5 test concentrations and a control). 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 colored 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 colored 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 temperated water bath and continuously illuminated at a mean light intensity of 8174 Lux, range 7350 8100 Lux. The light intensity was measured just before the start of the test below the coating cylinders in those areas, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (universal white L 25, 36 W) installed in top of the algal flasks.
The test duration was 72 hours.

TEST MEDIUM / WATER PARAMETERS see attachment


- Determination of cell concentrations: Samples of 1 ml test solution were taken out of all flasks under sterile conditions 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), three measurements per flask and time. In addition, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 32.0 mg/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:

EC10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 0.4 to 9.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 3.4 to 24.5

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

40.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 8.6 to 193.4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 1 to 26

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 8.8 to 74.4

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

127.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 18.6 to 880.1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 0.5 to 11

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 3.8 to 28

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

47.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 233

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 1.2 to 29.2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 81

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

134.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 19.7 to 917.4

Details on results:

All test media down to the lowest test concentration were little to strongly colored by the test substance.
In the control the cell density has increased from nominal 1x10E04 cells/ml at the start of the test (0 hours) to 68.06x10E04 cells/ml (mean value) after 72 hours by a factor of approximately 68. The biological results of both experimental parts A and B are identical. This is demonstrated by several calculated parameters, the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours and the EC values.
In both experimental parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 100 mg test substance/L. The NOEC (highest concentration tested without significant inhibition effect) amounted to 3.2 mg/L.
In all test concentrations the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. Specially in the test concentration range with significant growth inhibition effect (10 to 100 mg/L) the inhibition rates were in good conformity.
In all test concentrations the mean algal cell densities experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B (results of t-tests, two sided, p < 0 05).
Also at the EC values, calculated for both growth parameters, no significant differences were observable between the values in both experimental parts.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Probit analysis
Dunnett test one sided at 0.05
t-test at 0.05

Validity criteria fulfilled:

yes

Conclusions:

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

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. 201, adopted June 7, 1984. 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 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. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. 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 the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attachement section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attachement section 13

3. ANALOGUE APPROACH JUSTIFICATION
see attachement section 13

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 0.4 to 9.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 3.4 to 24.5

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

40.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 8.6 to 193.4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 1 to 26

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 8.8 to 74.4

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

127.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 18.6 to 880.1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 0.5 to 11

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 3.8 to 28

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

47.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 233

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 1.2 to 29.2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 81

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

134.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 19.7 to 917.4

Details on results:

All test media down to the lowest test concentration were little to strongly colored by the test substance.
In the control the cell density has increased from nominal 1x10E04 cells/ml at the start of the test (0 hours) to 68.06x10E04 cells/ml (mean value) after 72 hours by a factor of approximately 68. The biological results of both experimental parts A and B are identical. This is demonstrated by several calculated parameters, the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours and the EC values.
In both experimental parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 100 mg test substance/L. The NOEC (highest concentration tested without significant inhibition effect) amounted to 3.2 mg/L.
In all test concentrations the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. Specially in the test concentration range with significant growth inhibition effect (10 to 100 mg/L) the inhibition rates were in good conformity.
In all test concentrations the mean algal cell densities experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B (results of t-tests, two sided, p < 0 05).
Also at the EC values, calculated for both growth parameters, no significant differences were observable between the values in both experimental parts.

Reported statistics and error estimates:

Probit analysis
Dunnett test one sided at 0.05
t-test at 0.05

Validity criteria fulfilled:

yes

Conclusions:

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

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. 201, adopted June 7, 1984. 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 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. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. 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 the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nine concentrations of the test substance between nominal 100 and 0.39 mg/L, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only).

- Sampling method: The test samples at t=0 h above the working range were diluted with deionised water to obtain concentrations within the working range and analysed. The samples within and below the working range were analysed undiluted.
To verify the calculated concentrations below the validated working range, four standards were prepared (1n-4n), on the day of analysis, by weighing two amounts of the test substance, and dissolving it in defined volumes of the solvent, and injected once. These standards were compared with the calibration line of the working range.
The test samples at t=72 h above the working range were diluted with deionised water to obtain concentrations within the working range and analysed. The samples within and below the working range were analysed undiluted.

Vehicle:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC (American Type Culture Collection) 22662
- Source (laboratory, culture collection): LGC Promochem GmbH, Mercatorstraße 51, 46485 Wesel A Rhein, Germany
- Method of cultivation:
Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in 250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature of 23 ± 2°C under permanent light with an intensity of at least 6000 lux. In about weekly intervals 1 mL of the stock culture is diluted 100-fold with nutrient medium for precultivation and incubation is continued.
Precultures were prepared by incubating a new stock culture for four days. At the start of the experiment the cell density was determined and adjusted to about 100 000 algae/mL by diluting with nutrient medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 2 °C

pH:

The test substance only slightly altered the pH of the test media. The pH was between 7.14 and 7.45 at the start of the incubation in the test cultures and it was 7.19 in the control cultures. After 72 hours of incubation the pH was between 8.47 and 9.92 in the test cultures and it was 7.87 in the control cultures.
The pH in the control cultures changed by 0.68 units during the 72 hours of incubation, i.e. it was within the maximum change of 1.5 recommended by the guideline. Maximum pH changes of 2.64 in the algae cultures did not adversely affect the outcome of the study.

Nominal and measured concentrations:

Nominal concentrations: 0.391 mg/L; 0.781 mg/L; 1.56 mg/L; 3.13 mg/L; 6.25 mg/L; 12.5 mg/L; 25.0 mg/L; 50.0 mg/L; 100 mg/L.
Measured concentrations
0 hours: 0.16 mg/L, 0.455 mg/L, 1.1 mg/L, 2.485 mg/L, 5.335 mg/L, 10.71 mg/L, 22.275 mg/L, 44.175 mg/L, 91.15 mg/L
72 hours: < 0.003 mg/L, < 0.003 mg/L, < 0.003 mg/L, < 0.003 mg/L,< 0.003 mg/L; 0.41 mg/L, 2.92 mg/L, 2.65 mg/L, 29.33 mg/L
geometric mean: -, -, -, -, -, 2.083 mg/L, 8.065 mg/L, 10.809 mg/L, 51.705 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All test cultures and the blank were set up in 250 mL conical flasks (Erlenmeyer). The total culture volume was 30 mL in each case.
- Initial cells density: The complete test substance cultures consisted each of 3 mL inoculum (100 000 algae/mL) and of 27 mL of the respective test substance preparation.
- Control end cells density: The blanks without algae contained 27 mL of the respective test substance preparation and 3 mL of nutrient medium. The negative control cultures contained 3 mL of inoculum and 27 mL of nutrient medium.
- No. of organisms per vessel: 10 000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: All cultures and the blank were incubated at 23 ± 2 °C for three days in permanent light with an intensity of approximately 8000 lux measured at the level of the base plate of the conical flasks (wavelength 400–700 nm) while shaking.
- Light intensity and quality: As the test substance solutions were of intensive magenta colour, the test conditions were modified to decrease the influence of the colour on the growth inhibition. According to results of a preliminary test the light intensity was increased by reducing the distance between the light source and the algae cultures (approximately ½ of the usual distance to the light) and by reducing the light path within the cultures by reduction of the total culture volume (30 mL total culture volume instead of 100 mL).

EFFECT PARAMETERS MEASURED
- Observation intervals: 24 h, 48 h and 72 h after the onset of incubation
- Determination of cell concentrations: the cell densities of the algae were determined by a Casy Cell Counter, Model DT, Schärfe System GmbH, Germany (pore size 60 µm).
- Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined.
- The pH was determined in all test cultures, in the control and in the blank at the start of the exposure and 72 hours thereafter.
- The temperature of the incubation chamber was recorded in writing at the start of incubation and each time when cell counts were performed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0.391 mg/L; 0.781 mg/L; 1.56 mg/L; 3.13 mg/L; 6.25 mg/L; 12.5 mg/L; 25.0 mg/L; 50.0 mg/L; 100 mg/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 91 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 91 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

biomass

Details on results:

During the 72 hours incubation period the cell density in the control cultures was increased by a factor of about 149, corresponding to about 7.2 generations.
Compared to the negative controls, algal growth was moderately to slightly inhibited at all test substance concentrations: by 44.1 to 5.7 % based on the area under the growth curves or by 14.7 to 3.0 % based on the average growth rates.

At the start of the incubation period the actual test substance concentrations of the algal cultures were between 91.15 and 0.16 mg per L medium (84.58 mg/L in the blank without algae).

After 72 hours of incubation, the actual test substance concentrations were in the test cultures between 3.8 and 32.2 % of the actual starting concentrations (four highest concentrations, relevant for the determination of the EC50 values) or below the detection limit of the analytical method used (0.003 mg/L). In the blank without algae the actual test substance concentration was 39.7 % of the actual starting concentration.

According to the directive 92/69/EEC Part C.3, "the concentrations of the test substance shall be maintained to within 80 % of the initial concentrations throughout a time corresponding to the duration of the test". Due to the properties of the test substance this criterion was not met. This did not adversely affect the outcome of the study.

The results do not give a clear indication for an uptake of the test substance by the algae or for adherence of the test substance to them.

 

pH of the test cultures and of the controls

The test substance only slightly altered the pH of the test media. The pH was between 7.14 and 7.45 at the start of the incubation in the test cultures and it was 7.19 in the control cultures. After 72 hours of incubation the pH was between 8.47 and 9.92 in the test cultures and it was 7.87 in the control cultures.

The pH in the control cultures changed by 0.68 units during the 72 hours of incubation, i.e. it was within the maximum change of 1.5 recommended by the guideline. Maximum pH changes of 2.64 in the algae cultures did not adversely affect the outcome of the study.

Growth inhibition

Compared to the negative controls, algal growth was moderately to slightly inhibited at all test substance concentrations: by 44.1 to 5.7 % based on the area under the growth curves or by 14.7 to 3.0 % based on the average growth rates.

"No observed effect concentration" (NOEC)

As, during the incubation period of 72 hours, the four lowest actual test substance concentrations (relevant for the determination of the NOEC values) declined to below the limit of detection of the analytical method used (0.003 mg/L), the calculation/determination of the NOECs was based on the actual test substance concentrations determined at the start of the incubation (actual starting concentrations).

Two (identical) "no observed effect concentrations" (NOECs) were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the actual starting concentrations.

NOEC (0-72h) of BRF 112-1		Actual starting concentrations
based on the area under the growth curves		1.1 mg/L
based on the average growth rates		1.1 mg/L

 

 EC50-values

The calculation/determination of the EC values was based on the geometric means of the actual test substance concentrations determined at the start and at the end of the incubation and on the actual test substance concentrations determined at the start of the incubation (actual starting concentrations).

Two (identical) EC50 values were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the geometric means as well as by the actual starting concentrations.

EC50 (0-72h) of BRF 112-1	Actual starting concentrations	Geometric mean
based on the area under the growth curve:EbC50(0-72 h)	> 91 mg/L	> 52 mg/L
based on the average growth rates: ErC50(0-72 h)	> 91 mg/L	> 52 mg/L

Validity criteria fulfilled:

yes

Executive summary:

A Pseudokirchneriella subcapitata growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of BRF 112 -1 on the growth of a unicellular green algal species.

Test design

Nine concentrations of the test substance between nominal 100 and 0.39 mg/L, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only). A stock solution of the test substance was prepared by dilution with the culture medium (nutrient medium) and was further diluted to give the intended concentrations.

There were three replicates for each test and control culture. The cell count of the algae was about 10⁴cells/mL at the start of the exposure in each vessel.

In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the begin and at the end of the incubation period. Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined.

Possible test substance effects were determined by comparison of the areas under the growth curves and by comparison of the growth rates.

As the test substance solutions were of intensive magenta colour, the test conditions were modified to decrease the influence of the colour on the growth inhibition. According to results of a preliminary test the light intensity was increased by reducing the distance between the light source and the algae cultures and by reducing the light path within the cultures by reduction of the total culture volume.

Test substance concentrations

At the start of the incubation period the actual test substance concentrations of the algal cultures were between 91.15 and 0.16 mg per L medium (84.58 mg/L in the blank without algae).

After 72 hours of incubation, the actual test substance concentrations were in the test cultures between 3.8 and 32.2 % of the actual starting concentrations (four highest concentrations, relevant for the determination of the EC50 values) or below the detection limit of the analytical method used (0.003 mg/L). In the blank without algae the actual test substance concentration was 39.7 % of the actual starting concentration.

According to the directive 92/69/EEC Part C.3, "the concentrations of the test substance shall be maintained to within 80 % of the initial concentrations throughout a time corresponding to the duration of the test". Due to the properties of the test substance this criterion was not met. This did not adversely affect the outcome of the study.

The results do not give a clear indication for an uptake of the test substance by the algae or for adherence of the test substance to them.

pH

The test substance only slightly altered the pH of the test media. The pH was between 7.14 and 7.45 at the start of the incubation in the test cultures and it was 7.19 in the control cultures. After 72 hours of incubation the pH was between 8.47 and 9.92 in the test cultures and it was 7.87 in the control cultures.

The pH in the control cultures changed by 0.68 units during the 72 hours of incubation, i.e. it was within the maximum change of 1.5 recommended by the guideline. Maximum pH changes of 2.64 in the algae cultures did not adversely affect the outcome of the study.

Increase in cell densities

During the 72 hours incubation period the cell density in the control cultures was increased by a factor of about 149, corresponding to about 7.2 generations.

Growth inhibition

Compared to the negative controls, algal growth was moderately to slightly inhibited at all test substance concentrations: by 44.1 to 5.7 % based on the area under the growth curves or by 14.7 to 3.0 % based on the average growth rates.

NOEC and EC50-values

As, during the incubation period of 72 hours, the four lowest actual test substance concentrations (relevant for the determination of the NOEC values) declined to below the limit of detection of the analytical method used (0.003 mg/L), the calculation/determination of the NOECs was based on the actual test substance concentrations determined at the start of the incubation (actual starting concentrations) and the EC50 values were based on the geometric means of the actual test substance concentrations determined at the start and at the end of incubation and on the actual starting concentrations.

Two (identical) "no observed effect concentrations" (NOECs) were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the actual starting concentrations.

NOEC (0-72h) of BRF 112-1	Actual starting concentrations
based on the area under the growth curves	1.1 mg/L
based on the average growth rates	1.1 mg/L

 

Two (identical) EC50 values were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the geometric means as well as by the actual starting concentrations.

EC50 (0-72h) of BRF 112-1	Actual starting concentrations	Geometric mean
based on the area under the growth curve:EbC50(0-72 h)	> 91 mg/L	> 52 mg/L
based on the average growth rates: ErC50(0-72 h)	> 91 mg/L	> 52 mg/L

Description of key information

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance on algae was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Additional information

A Pseudokirchneriella subcapitata growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of Reaktiv Rot F66813 on the growth of a unicellular green algal species.

Nine concentrations of the test substance between nominal 100 and 0.39 mg/L, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only). A stock solution of the test substance was prepared by dilution with the culture medium (nutrient medium) and was further diluted to give the intended concentrations. There were three replicates for each test and control culture. The cell count of the algae was about 10⁴ cells/mL at the start of the exposure in each vessel. In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the begin and at the end of the incubation period. Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined. Possible test substance effects were determined by comparison of the areas under the growth curves and by comparison of the growth rates.

As the test substance solutions were of intensive magenta colour, the test conditions were modified to decrease the influence of the colour on the growth inhibition. According to results of a preliminary test the light intensity was increased by reducing the distance between the light source and the algae cultures and by reducing the light path within the cultures by reduction of the total culture volume.

At the start of the incubation period the measured test substance concentrations of the algal cultures were between 91.15 and 0.16 mg/L medium (84.58 mg/L in the blank without algae). After 72 hours of incubation, the actual test substance concentrations were in the test cultures between 3.8 and 32.2 % of the actual starting concentrations (four highest concentrations, relevant for the determination of the EC50 values) or below the detection limit of the analytical method used (0.003 mg/L). In the blank without algae the actual test substance concentration was 39.7 % of the actual starting concentration.

This difference to the nominal test substance concentrations is due to the fact that only the sulfatoethylsulfone-form of the dye was analytically quantified and not its hydrolysed forms the vinyl sulfone, which is the actual active dye-form or its final hydrolysis product, the hydroxyethylsulfone. As shown in the abiotic degradation studies, the test substance hydrolyses rapidly to its vinyl sulfone, and finally the hydroxyethylsulfone-form.Both modification are coloured and have similar ecotoxicological properties as the sulfatoethylsulfone. The analytical verification of the test substance concentration in the fish and daphnia studies over 96 and 48 hours, respectively, which were done by VIS-spectrophotometric determination proved a stable dye content of the coloured components over the entire testing period. Hence, for the effect concentration it is justified to use the nominal concentrations.

The results do not give a clear indication for an uptake of the test substance by the algae or for adherence of the test substance to them.

The test substance only slightly altered the pH of the test media. The pH was between 7.14 and 7.45 at the start of the incubation in the test cultures and it was 7.19 in the control cultures. After 72 hours of incubation the pH was between 8.47 and 9.92 in the test cultures and it was 7.87 in the control cultures. The pH in the control cultures changed by 0.68 units during the 72 hours of incubation, i.e. it was within the maximum change of 1.5 recommended by the guideline. Maximum pH changes of 2.64 in the algae cultures did not adversely affect the outcome of the study.

During the 72 hours incubation period the cell density in the control cultures was increased by a factor of about 149, corresponding to about 7.2 generations. Compared to the negative controls, algal growth was moderately to slightly inhibited at all test substance concentrations: by 44.1 to 5.7 % based on the area under the growth curves or by 14.7 to 3.0 % based on the average growth rates.

Two (identical) "no observed effect concentrations" (NOECs) were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the nominal starting concentrations.

The NOEC (0-72h) of the test substance is 1.6 mg/L based on the area under the growth curves and based on the average growth rates.

Likewise, two (identical) EC₅₀values were derived – based on the area under the growth curves and based on the average growth rates calculated/determined by the geometric means as well as by the actual starting concentrations.

The EC₅₀(0-72h) of the test substance is > 100 mg/L based on the area under the growth curves (E_bC₅₀(0-72h))

and based on the average growth rates (E_rC₅₀(0-72h)).

Dyes that lead to a heavily coloured test medium are always a challenge for the algae test. The OECD Series on Testing and Assessment Number 23. “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”, states the following for algae tests with coloured substances.

Coloured substances can absorb photosynthetically active light and hence limit growth of algal cultures. Absorption will be proportional to test substance concentration and as a consequence it can result in growth inhibition which is difficult to distinguish from inherent toxicity.

The need for clear guidance on how to determine the inherent toxicity of coloured substances to algae is widely recognised (EC, 1996a) and work is ongoing in this area (Justesen and Nyholm, 1998).

The International Organization for Standardization (ISO, 1997) has identified the following general strategies for discriminating between effects resulting from toxicity and light absorption:

1. testing at different light intensities, since the growth rate above light saturation is almost independent from the light intensity;
2. reducing the light path by reducing the depth or the volume;
3. measuring the reduction of cell proliferation in a control batch when the light previously passes through a liquid light transmission filter, e.g. a flat dish containing corresponding dilutions of the sample with the corresponding colour and layer thickness; and
4. utilising a light transmission filter by preparing reciprocal dilutions to the exposure concentrations thus maintaining a common light path length (constant depth) and the spectral absorbance characteristics of the sample.

However, experience with testing of Reactive Dyes which lead to an intense staining of the test medium showed that the first two recommendations are not able to completely compensate the light absorption of the dye. The test method, which provide the best results for these dyes are the ones which used the same concentrations of the test substance as a filter placed above the test medium with the algae without the test substance (method 3). This testing method was used with a structural analogue of Reaktiv Rot F66813 and is described in the following, demonstating unequivocally that all effects observed on algae growth were solely due to light absorption effects.

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. 201, adopted June 7, 1984. 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 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. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significantly different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. 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 the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.