Bis[N-(4-{[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-ethylethanaminium] [mono and bis(dodecanyl, branched)]-(sulfonatophenoxy) benzenesulfonate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrakis(4-{[4-(diethylamino)phenyl][4-(ethylamino)naphthalen-1-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1-iminium) 2-(dodecan-5-yl)-4-(4-sulfonatophenoxy)benzene-1-sulfonate 2-(dodecan-5-yl)-4-[3-(dodecan-5-yl)-4-sulfonatophenoxy]benzene-1-sulfonate

EC Number:

700-579-6

Molecular formula:

C90H114O7S2N6 (MW = 1454) and C102H138O7S2N6 (MW = 1622)

IUPAC Name:

tetrakis(4-{[4-(diethylamino)phenyl][4-(ethylamino)naphthalen-1-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1-iminium) 2-(dodecan-5-yl)-4-(4-sulfonatophenoxy)benzene-1-sulfonate 2-(dodecan-5-yl)-4-[3-(dodecan-5-yl)-4-sulfonatophenoxy]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Batch number: 310551

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 14 ml of test item diluted in 1 L liters of ultra-pure water (stock solution).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not mentionned

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Age of inoculum (at test initiation): 3 days

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22°C +/2

pH:

8.1 à 9.1

Nominal and measured concentrations:

Nominal concentration:
0-7.0-8.4-9.8-11.2-12.6 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer
- Material, size, headspace, fill volume: Erlenmeyer with 60 mL of the tested solution
- Initial cells density: 1000 cells/mL
- No. of vessels per concentration : 3
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality:4500 lux
- stirring: 250 min-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Mallassez counting chamber


TEST CONCENTRATIONS
- the maximum tested concentration was 12,6 mg/L. According to the maximum water solubility (14 mg/l) and the quantification limit of the algal growth inhibition test: 90 % of the stock solution, the maximal tested concentration was 12.6 mg/L.
A stock solution at 14 mg/L was prepared with pure water. Then a feeding media was added so as to represent 10% of the final solution.
Therefore the highest concentration was 12,6 mg/L (0.9 x 14)

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no mentionned
- the results correspond to the maximal concentration tested

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 (72h): 1.21 mg/L (belongs to the recommended range of values: 0.92 - 1.46 mg/L)

Reported statistics and error estimates:

CE x % calculated using the Hill equation (macro Regtox_ev6.6.2xls)
NOEC calculated with the T-test of Bonferroni (Toxcalc software)

Any other information on results incl. tables

Results at t = 24h, 48h and 72h:

 

t = 24 h
Concentration (mg/L)	Cells Concentration x 10^6
	1	2	3	4	5	6	Average Cells number	Cells Inhibition
0	0.075	0.073	0.076	0.079	0.077	0.079	0.08
7.0	0.071	0.082	0.079				0.077	-1%
8.4	0.072	0.077	0.068				0.072	6%
9.8	0.074	0.078	0.077				0.076	0%
11.2	0.068	0.076	0.057				0.067	12%
12.6	0.056	0.067	0.074	0.056	0.073	0.077	0.067	12%

 

 

t = 24 h
Concentration (mg/L)	Growth Rate
	1	2	3	4	5	6	Average Cells number	Growth rate Inhibition
0	2.01	1.99	2.03	2.07	2.05	2.07	2.04
7.0	1.96	2.11	2.06				2.04	0%
8.4	1.97	2.04	1.91				1.97	3%
9.8	2.00	2.06	2.04				2.03	0%
11.2	1.92	2.03	1.74				1.90	7%
12.6	1.72	1.90	2.00	1.73	1.99	2.05	1.90	7%

 

 

t = 48 h
Concentration (mg/L)	Cells Concentration x 10^6
	1	2	3	4	5	6	Average Cells number	Cells Inhibition
0	0.57	0.64	0.64	0.71	0.65	0.62	0.64
7.0	0.54	0.65	0.58				0.59	7%
8.4	0.63	0.77	0.56				0.65	-2%
9.8	0.60	0.59	0.62				0.60	5%
11.2	0.431	0.477	0.587				0.50	22%
12.6	0.55	0.44	0.37	0.52	0.55	0.60	0.50	21%

 

t = 48 h
Concentration (mg/L)	Growth Rate
	1	2	3	4	5	6	Average Cells number	Growth rate Inhibition
0	2.02	2.08	2.08	2.13	2.09	2.06	2.08
7.0	2.00	2.09	2.03				2.04	2%
8.4	2.07	2.17	2.01				2.08	0%
9.8	2.05	2.04	2.06				2.05	1%
11.2	1.88	1.93	2.04				1.95	6%
12.6	2.01	1.89	1.80	1.98	2.00	2.05	1.95	6%

 

 

 

 

t = 72 h
Concentration (mg/L)	Cells Concentration x 10^6
	1	2	3	4	5	6	Average Cells Number	Cells Inhibition
0	3.54	3.70	4.12	3.83	3.61	3.60	3.73
7.0	3.77	3.95	3.85				3.86	-3%
8.4	4.02	4.25	3.97				4.08	-9%
9.8	3.25	3.20	3.89				3.45	8%
11.2	3.54	3.82	4.02				3.79	-2%
12.6	3.75	3.39	3.22	4.36	3.76	3.84	3.72	0%

 

 

t = 72 h
Concentration (mg/L)	Growth Rate
	1	2	3	4	5	6	Average Cells number	Growth rate Inhibition	Standard deviation	Variation coefficient (%)
0	1.96	1.97	2.01	1.98	1.96	1.96	1.97		0.017	0.86
7.0	1.98	1.99	1.98				1.98	-1%	0.006	0.31
8.4	2.00	2.02	2.00				2.00	-2%	0.010	0.49
9.8	1.93	1.92	1.99				1.95	1%	0.029	1.51
11.2	1.96	1.98	2.00				1.98	0%	0.017	0.88
12.6	1.98	1.94	1.92	2.03	1.98	1.98	1.97	0%	0.032	1.63

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions:
CE 50r - 72h > 12.6 mg/L
NOEC - 72 h > 12.6 mg/L

Executive summary:

The acute algal toxicity test for the substance Sepisol Fast Blue 85219 was carried out to determine the ecotoxicological profile of the test substance against an unicellular green algae. As test organism Pseudokirchnerella subcapitata was used and the NF EN ISO 8692 (2005) standard method was followed.

 

The parameters which were to be determined were the effective concentrations (EC) and the no observed effect concentration 72h (NOEC-72h) values with regard to the growth rate (ErC).

The following nominal concentrations were tested: 0; 7.0; 8,4; 9,8; 11,2 and 12,6 mg/L. The maximum tested concentration was 12,6 mg/L. According to the maximum water solubility and the quantification limit of the algal growth inhibition test: 90 % of the stock solution, the maximal tested concentration was determined to be 12.6 mg/L.

A stock solution was prepared by diluting 14 mg of test material in 1 liter of pure water.

Each concentration and the controls were inoculated with approximately 10 000 algae per mL.

The test was carried out over 72 hours at a temperature of 22°C +/- 2 and constantly stirred (250 min^-1). Each vessel concentration was tested 3 times. 5 controls were run with the same experimental conditions. Algal concentration was counted using a Mallassez counting chamber. Once a day the extinction of an aliquot of the test-vessels (incubation) was measured.

Validation experiments and the precultivation ensured that the algal were of appropriate vialibilty and susceptibility.

The results of this study showed, that the substance Sepisol Fast Blue 85219 did not inhibit cells nor reduce the growth rate of the test organism at 12, 6 mg/L. The quantification limit of the algal growth inhibition test being 90 % of the stock solution, the maximal tested concentration was 12.6 mg/L.

The ErC 10 (72h) and the NOEC (72h) were thus determined to be higher than 12.6 mg/L (with 12.6 being the quantification limit of the inhibition test: 90 % and the maximum water solubity being 14 mg/L).