Bis{tris[4-(mono and dimethylamino)phenyl]methylium} 2-(bis{4-[ethyl(3-sulfonatobenzyl)amino]phenyl}methyliumyl)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:

2008/06/10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 2 because the study followed a standatd guideline of reference (NF EN ISO 8672, 2005), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid. However the study was not conducted under GLP conditions.

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:

Bis{tris[4-(mono and dimethylamino)phenyl]methylium} 2-(bis{4-[ethyl(3- sulfonatobenzyl)amino]phenyl}methyliumyl)benzenesulfonate

IUPAC Name:

Bis{tris[4-(mono and dimethylamino)phenyl]methylium} 2-(bis{4-[ethyl(3- sulfonatobenzyl)amino]phenyl}methyliumyl)benzenesulfonate

Constituent 2

Reference substance name:

Bis{tris[4-(mono and dimethylamino)phenyl]methylium} 2-(bis{4-[ethyl(3-sulfonatobenzyl)amino]phenyl}methyliumyl)benzenesulfonate

EC Number:

700-610-3

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

Bis{tris[4-(mono and dimethylamino)phenyl]methylium} 2-(bis{4-[ethyl(3-sulfonatobenzyl)amino]phenyl}methyliumyl)benzenesulfonate

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Blue 2BR
- Substance type: Organic salt
- Physical state: dark blue powder
- Lot/batch No.: 905536
- Stability under test conditions: Stable
- Storage condition of test material: at ambient temperature

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): 73 mg/l and corresponding pH = 3,8 (with pure water)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72

Test conditions

Test temperature:

22 +/- 2°C

pH:

From 8.1 to 8.2 at To
From 8.4 to 9.3 at T72h

Nominal and measured concentrations:

2.6 - 1.8 - 1.2 - 0.95 - 0.66 and 0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer
- Material, size, headspace, fill volume: Erlenmeyer with 60 mL of the test item at diverse concentrations.
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: 4500 lux
- Stirring: 250 rev/min

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: square root of 2
- Test concentrations: 2.6 - 1.8 - 1.2 - 0.95 - 0.66 and 0 mg

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72

Dose descriptor:

EC10

Effect conc.:

1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.92 - 1.1 mg/L

Effect concentrations 2

Duration:

72

Dose descriptor:

EC20

Effect conc.:

1.3

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.2 - 1.4 mg/L

Effect concentrations 3

Duration:

72

Dose descriptor:

EC50

Effect conc.:

1.8

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.8 - 1.9 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.66

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

CEx% calculated using the Hill Equation (macro Regtox_ev 6.6.2xls)
NOEC calculated using the T-test of Bonferroni (Toxcalc software)

Any other information on results incl. tables

Results at 72h, 48h and 24h:

t = 72 h
Concentration mg/L	Cells concentration x 10^6
	1	2	3	4	5	6	Average cells number	Cells inhibition
0	3.95	3.82	4.08	3.76	4.14	3.50	3.87
0.66	3.55	3.73	3.95				3.74	3%
0.95	1.83	1.79	1.85				1.82	53%
1.2	1.05	1.08	0.99				1.04	73%
1.8	0.31	0.30	0.26				0.29	93%
2.6	0.028	0.024	0.035				0.03	99%
	Growth Rate
	1	2	3	4	5	6	Average cells number	Growth rate inhibition
0	1.99	1.98	2.00	1.98	2.01	1.95	1.99
0.66	1.96	1.97	1.99				1.97	1%
0.95	1.74	1.73	1.74				1.73	13%
1.2	1.55	1.56	1.53				1.55	22%
1.8	1.15	1.13	1.08				1.12	44%
2.6	0.34	0.29	0.42				0.35	82%

 

 

t = 48 h
Concentration mg/L	Cells concentration x 10^6
	1	2	3	4	5	6	Average cells number	Cells inhibition
0	0.67	0.55	0.50	0.51	0.68	0.67	0.60
0.66	0.51	0.52	0.54				0.52	12%
0.95	0.46	0.45	0.43				0.45	25%
1.2	0.18	0.17	0.18				0.18	71%
1.8	0.068	0.061	0.049				0.06	90%
2.6	0.01	0.01	0.02				0.01	98%
	Growth Rate
	1	2	3	4	5	6	Average cells number	Growth rate inhibition
0	2.10	2.00	1.95	1.97	2.11	2.10	2.04
0.66	1.97	1.97	2.00				1.98	3%
0.95	1.92	1.90	1.88				1.90	7%
1.2	1.43	1.43	1.44				1.43	30%
1.8	0.96	0.90	0.80				0.89	57%
2.6	0.18	0.00	0.29				0.16	92%

 

 

 

t = 24 h
Concentration mg/L	Cells concentration x 10^6
	1	2	3	4	5	6	Average cells number	Cells inhibition
0	0.083	0.083	0.079	0.073	0.070	0.069	0.08
0.66	0.059	0.064	0.062				0.061	20%
0.95	0.050	0.029	0.053				0.051	33%
1.2	0.026	0.045	0.027				0.027	64%
1.8	0.019	0.017	0.020				0.019	75%
2.6	0.012	0.012	0.014				0.013	84%
	Growth Rate
	1	2	3	4	5	6	Average cells number	Growth rate inhibition
0	2.12	2.11	2.07	1.99	1.94	1.93	2.03
0.66	1.77	1.85	1.82				1.81	11%
0.95	1.61	1.60	1.67				1.63	20%
1.2	0.96	1.07	1.00				1.01	50%
1.8	0.65	0.53	0.69				0.63	69%
2.6	0.15	0.18	0.34				0.22	89%

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental condition: growth rate inhibition
* 72h - EC10 = 1.0 mg/L (0.92 - 1.1 mg/L)
* 72h - EC20 = 1.3 mg/L (1.2 - 1.4 mg/L)
* 72h - EC50 = 1.8 mg/L (1.8 - 1.9 mg/L)
* 72h NOEC = 0.66 mg/L

Executive summary:

The acute algal toxicity test for the substance Sepisol Fast Blue 2BR was carried out to determine the ecotoxicological profile of the test substance against an unicellular green algae. The test organism Pseudokirchnerella subcapitata was used and the NF EN ISO 8692 (2005) standard method was followed.

The effective concentrations (EC) and the non-observed effect concentration 72h (72h-NOEC) values with regard to the growth rate (ErC) were determined.

The following nominal concentrations were tested: 2.6 - 1.8 - 1.2 - 0.95 - 0.66 and 0 mg/L. The maximum tested concentrations was 2.6 mg/L.

A stock solution was prepared by diluting 73 mg of test material in 1 liter of pure water

Each concentration and the controls were inoculated with approximately 10 000 cells per mL. The test was carried out over 72 hours at a temperature of 22°C+/- 2°C and constantly stirred (250 rev/min).

Each vessel concentration was tested 3 times. 6 controls were run with the same experimental condition. 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 vialibility and susceptibility.

The results of this study showed, that the substance Sepisol Fast Blue 2BR inhibit cells and reduce the growth rate of the test organism at the tested concentrations. Under experimental conditions, the following parameters were determined:

* 72h - EC10 = 1.0 mg/L (0.92 - 1.1 mg/L)

* 72h - EC20 = 1.3 mg/L (1.2 - 1.4 mg/L)

* 72h - EC50 = 1.8 mg/L (1.8 - 1.9 mg/L)

* 72h NOEC = 0.66 mg/L