7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

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:

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

Adequacy of study:

key study

Justification for type of information:

Please refer section 13 for read across justification.

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

no

Details on sampling:

On request of the sponsor no analytical measurements have been performed, since the biological results of the experimental part A in this test are in conformity with the biological results of earlier tests. Therefore, all reported results are related to the nominal concentrations of the test substance.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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: 3.2, 10.0, 32.0, 100 and 320 mg test substance/L. Additionally, a control (test water without addition of the test substance) was tested.
- Controls: Blank control performed with the test solution without test substance.
- Evidence of undissolved material: no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Algae (Scenedesmus subspicatus CHODAT)
- Strain: strain N° 86.81 SAG
- Source: Collection of algae cultures from Institute of Plant Physiology at the University of Göttingen, D-37073 Göttingen.
- Method of cultivation: The algae were cultivated and tested in synthetic test water, prepared according to the OECD Guideline No. 201 (1981)

ACCLIMATION
- Acclimation conditions: same as the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 0.2 °C

pH:

pH at the start of the test: 8
pH at the end of the test: 10.5

Salinity:

-

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 3.2, 10.0, 32.0, 100 and 320 mg/L
Measured concentrations: No concentrations of the test substance were measured.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flaks 100 mL
- Type: closed, The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.
- Material: 50 mL algal suspension, continuously stirred by magnetic stirrers
- Aeration: no
- Initial cells density: 10.000 cells per mL test solution.
- Control end cells density: 77.89E4 cells/mL (mean value) after 72 hours.
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
In deionized water salts analytical grade were dissolved to following final nominal concentrations:
Macro-nutrients:
NaHCO3: 50.0 mg/L
CaCl2 * 2H2O: 18.0 mg/L
NH4Cl: 15.0 mg/L
MgSO4 * 7H2O 15.0 mg/L
MgCl2 * 6 H2O: 12.0 mg/L
KH2PO4: 1.6 mg/L

Trace elements:
Na2EDTA * 2 H2O: 100.0 µg/L
FeCl3 * 6 H2O: 80.0 µg/L
MnCl2 * 4 H2O: 415.0 µg/L
H3BO3: 185.0 µg/L
Na2MoO4 * 2 H2O: 7.0 µg/L
ZnCl2: 3.0 µg/L
CoCl2 * 6 H2O: 1.5 µg/L
CuCl2 * 2 H2O: 0.01 µg/L

- Conductivity: < 0.1 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated
- Light intensity and quality: 8147 Lux, range 7600 - 8530 Lux by fluorescent tubes

EFFECT PARAMETERS MEASURED: The algae cell densities in the samples were determined by counting with an electronical particle counter after 24, 48 and 72 hours.

RANGE FINDING TEST
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 and these results are not mentioned in the report, but the raw data of the range-finding test will be archived under the RCC Project number of the present study.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: real toxic effect.

Details on results:

- Observation of abnormalities: A slightly higher inhibition effect on the algal growth was observed in all test concentrations.
- Effect concentrations exceeding solubility of substance in test medium: no
The ErC 50 and EbC50 for Remazol Brillantorange 3R Oxyfarbstoff could be determined to be clearly higher than 320 mg test substance/L when based on the real toxic effect and the NOEC was determined to be ca. 3.2 mg/L.

Results with reference substance (positive control):

No test with reference substance was performed.

Reported statistics and error estimates:

The corresponding EC10- and EC90-values and their 95%-confidence limits were calculated by Probit Analysis. For the determination of the NOEC/LOEC, the calculated mean growth rates at the test concentrations were tested on significant differences to the control value by the Dunnet-Test.

All test media down to the lowest test concentration of nominal 3.2 mg/L were little to strongly coloured by the test substance.

Slightly higher inhibition effect on the algal growth was observed in all test concentrations 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 inexperimental part B were higher than the corresponding EC-values in experimental part A.

 

Also the determined NOEC/LOEC-values in experimental part A were lower than in experimental part B: in experimental part A the NOEC (highest concentration tested without toxic effects) was determined at nominal 3.2 mg test substance/l, the LOEC at nominal 10 mg test substance/L. In comparison, in experimental part B the NOEC was determined at nominal 10.0 mg test substance/L, the LOEC at nominal 32 mg test substance/L (since the growth rate r was statistically significantly lower than in control during the test period of 72 hours first at this test concentration).

For the quantification of the algicidal effect versus the growth inhibition due to reduced light intensities, 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 7.2 - 31.2 %, for the growth rate r between 2.2 and 19.4 %. These 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 filter effect of the dyestuff, the real toxic effect on the growth of Scenedesmus subspicatus after the 72 hours exposure period amounted to lower than 50 % up to the highest test concentration of 320 mg test substance/L. And more than 50 % of the inhibition effect, determined in experimental part A (as in a usual algal growth inhibition test) was caused only by the 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 growing in the test concentration of nominal 320 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 high concentration of dissolved test substance could not be observed.

In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance Remazol-Brillantorange 3R Oxyfarbstoff on Scenedesmus subspicatus was caused in a high degree (> 50 %) due to the indirect effect, the Tight absorption in the coloured test solutions. Thus, the 72-hours ErC 50 and EbC50 could be determined to be clearly higher than 320 mg test substance/L when based on the real toxic effect and the NOEC was determined to be ca. 3.2 mg/L.

Table 1:pH-values during the test period

Nominal test subst. conc. (mg/l)	pH-values
	Start	End
control	8	10.5
3.2	8	10.3
10	7.9	9.5
32	7.8	9
100	7.7	8.6
320	7.6	8.4

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance reactive orange on Scenedesmus subspîcatus was caused in a high degree (> 50 %) due to the indirect effect, the light absorption in the coloured test solutions. Thus, the 72-hours ErC50 and EbC 50 for Remazol Brillantorange 3R Oxyfarbstoff could be determined to be clearly higher than 320 mg test substance/L when based on the real toxic effect and the NOEC was determined to be ca. 3.2 mg/L.

Executive summary:

The influence of the test substance reactive 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 tight 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 tight conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 3.2, 10.0, 32.0, 100 and 320 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 all test concentrations in experimental part A, where the algae grew in the test solutions with dissolved test sub-stance 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 higher than the corresponding EC-values in experimental part A (see below).

Also the determined NOEC/LOEC-values in experimental part A were lower than in experimental part B: in experimental part A the NOEC (highest concentration tested without toxic effects) was determined at nominal 3.2 mg test substance/L, the LOEC (lowest concentration tested with toxic effects) at nominal 10 mg test substance/L. In comparison, in experimental part B the NOEC was determined at nominal 10.0 mg test substance/L, the LOEC at nominal 32 mg test substance/L.

 

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

Experimental part A

- EbC 50 (0 - 72 h) 12.4 mg test substance/L (95 % conf. Limits) (1.9 - 81.1 mg test substance/L)

- EbC 10 (0 - 72 h) 0.5 mg test substance/L  (95 % conf. Limits   0.0 - 55.7 mg test substance/L)

- EbC 90 (0 - 72 h) 291.0 mg test substance/L (95 % conf. limits12.4 mg test substance/L

10.7 - 7896         mg test substance/l)

 

Experimental part B

- EbC 50 (0 - 72 h) 60.0 mg test substance/l (95 % conf. limits   13.2 - 271.9 mg test substance/l)

- EbC 10 (0 - 72 h) 4.3 mg test substance/L (95 % conf. limits     0.3 -62.2 mg test substance/l)

- EbC 90 (0 - 72 h) 828.2 mg test substance/l (95 % conf. limits not calculable)

Growth rate  r in:

 

Experimental part A

-ErC 50 (0 - 72 h) 219.4 mg test substance/L (95 % conf. Limits 11.2 - 4278         mg test substance/L)

-ErC 10 (0 - 72 h) 5.0 mg test substance/L (95 % conf. Limits 0.1 -212.4 mg test substance/L)

-ErC 90 (0 - 72 h) 9568 mg test substance/L (95 % conf. Limits not calculable)

 

Experimental part B

- ErC 50 (0 - 72 h) 675.7 mg test substance/L* (95 % conf. Limits 11.2 – 4278 mg test substance/L)

- ErC 10 (0 - 72 h) 28.7 mg test substance/L  (95 % conf, limits    13.4 – 33947 mg test substance/L)

-ErC 90 (0 - 72 h) 15924 mg test substance/L* (95 % conf. Limits not calculable)

* this EC-value should be taken with caution, because inhibition of }t exceeds in none of the tested concentrations 50%

 

For the quantification of the algicidal effect versus the growth inhibition due to reduced light intensities, 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 7.2 - 31.2 %, for the growth rate r between 2.2 and 19.4 %.

 

These differences between the inhibition rates in parts A and B were obviously caused by a real toxic effect of the test sub-stance on the algal growth. Thus, after correction of the total inhibition effect by the filter effect of the dyestuff, the real toxic effect of the test substance on the growth of Scenedesmus subspicatus after the 72 hours exposure period amounted to lower than 50 % up to the highest test concentration of 320 mg test substance/L. And more than 50 % of the inhibition effect, determined in experimental part A (as in a usual algal growth inhibition test) was caused only by the filter effect of the dyestuff.

The shape of the algal cells, growing in the test solutions with the dissolved test substance at the highest test concentration of nominal 320 mg test substance/l was not modified compared to the cells in the control.

 

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 (> 50 %) due to the indirect effect, the light absorption in the coloured test solutions. Thus, the 72-hours ErC 50 and EbC50 could be determined to be clearly higher than 320 mg test substance/L when based on the real toxic effect and the NOEC was determined to be ca. 3.2 mg/L.

On request of the sponsor no analytical measurements have been performed, since the biological results of the experimental part A in this test are in conformity with the biological results of earlier tests. Therefore, all reported results are related to the nominal concentrations of the test substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer section 13 for read across justification.

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

no

Details on sampling:

- No concentrations of the test substance were measured.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were performed by dilution of the stock solution with destilled water.
- Controls: Blank control performed with the test solution without test substance.
- Evidence of undissolved material: no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Algae (Scenedesmus subspicatus Chodat)
- Strain: 8681
- Source: SAG
- Method of cultivation:according to OECD guideline

ACCLIMATION
- Acclimation period: same as the test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

20 °C

pH:

7 ± 1

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 5 and 10 mg/L
- Measured concentrations: No concentrations of the test substance were measured.

Details on test conditions:

TEST SYSTEM
- Initial cells density: 4.58 x 10^7 cell/h
- Control end cells density: yes, by fluorescein
- No. of vessels per concentration: 1
- No. of vessels per control (negative): 1

TEST MEDIUM / WATER PARAMETERS
- Conductivity: > 1.4 10^7 Ohm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: according to OECD guideline
- Light intensity and quality: according to OECD guideline

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: not observed
- Unusual cell shape: not observed
- Colour differences: not observed
- Flocculation: not observed
- Adherence to test vessels: not observed
- Aggregation of algal cells: not observed
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
After 72 h the EC10 and the EC50 were determined to be > 10 mg/L.

Results with reference substance (positive control):

No test with reference substance were performed.

Table 1: Inhibition of the growth rate

test concentrations (mg/L)	growth rate (cell/h)	inhibition %
0	31803
5	40747.9	0
10	40369.6	0

Validity criteria fulfilled:

yes

Conclusions:

After 72 h the EC10 and the EC50 were determined to be > 10 mg/L.

Executive summary:

The test substance was tested for toxicity about algal growth inhibition in accordance to OECD 201 with Scenedesmus subspicatus Chodat after 24,48 and 72 hours of test period. The substance does not show any form of inhibition at the highest concentration.

Following results were determined after 72 hours:

EbC10 > 10 mg/L                

ErC10 > 10 mg/L

EbC50 > 10 mg/L

ErC50 > 10 mg/L

Description of key information

After 72 hours the EC50 was determined to be greater than 320 mg/L (nominal value) and the NOEC was determined to be 3.2 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

The influence of the test substance structural analogue 01 -OH 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 tight 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 tight conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 3.2, 10.0, 32.0, 100 and 320 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 all test concentrations in experimental part A, where the algae grew in the test solutions with dissolved test sub-stance 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 higher than the corresponding EC-values in experimental part A.

Also the determined NOEC/LOEC-values in experimental part A were lower than in experimental part B: in experimental part A the NOEC (highest concentration tested without toxic effects) was determined at nominal 3.2 mg test substance/L, the LOEC (lowest concentration tested with toxic effects) at nominal 10 mg test substance/L. In comparison, in experimental part B the NOEC was determined at nominal 10.0 mg test substance/L, the LOEC at nominal 32 mg test substance/L.

 

For the quantification of the algicidal effect versus the growth inhibition due to reduced light intensities, 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 7.2 - 31.2 %, for the growth rate r between 2.2 and 19.4 %.

Concentration	% inhibition growth rate after 72 hours
(mg/L)	part A	part B	A -B
3.2	4.3	2.1	2.2
10	16.5	0.3	16.2
32	31.6	14.1	17.5
100	37.3	26.6	10.7
320	53.1	33.7	19.4

These differences between the inhibition rates in parts A and B were obviously caused by a real toxic effect of the test sub-stance on the algal growth. Thus, after correction of the total inhibition effect by the filter effect of the dyestuff, the real toxic effect of the test substance on the growth of Scenedesmus subspicatus after the 72 hours exposure period amounted to lower than 50 % up to the highest test concentration of 320 mg test substance/L. And more than 50 % of the inhibition effect, determined in experimental part A (as in a usual algal growth inhibition test) was caused only by the filter effect of the dyestuff.

The shape of the algal cells, growing in the test solutions with the dissolved test substance at the highest test concentration of nominal 320 mg test substance/l was not modified compared to the cells in the control.

 

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 (> 50 %) due to the indirect effect, the light absorption in the coloured test solutions. Thus, the 72-hours ErC 50 and EbC50 could be determined to be clearly higher than 320 mg test substance/L when based on the real toxic effect and the NOEC was determined to be ca. 3.2 mg/L.