Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

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:

From 2011-01-25 to 2011-02-04

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliabitity is rated 3 because the study followed a standard guideline of reference (OECD 201), which describes a procedure designed to evaluate this endpoint. But the results were reviewed and not considered as valid. Furthermore the study was not conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

EC Number:

700-615-0

Molecular formula:

BIS: C82H74N14CuS2O6 TRIS: C107H103N17CuS3O9 TETRA: C132H132N20CuS4O12

IUPAC Name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Violet 3B
- Substance type: Organocopper salt
- Lot/batch No.: 028942

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: 2 mg/L

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain:CCAP278/2
- Source (laboratory, culture collection): SAMS research Services, Ltd, Scotland
- Age of inoculum (at test initiation): culture in exponential growth phase
- Method of cultivation: Culture medium : LC-Oligo (pH 7,1 +/- 0,1). Temperature of 21-24 °C. 16h light/8h dark (around 4500 lux). Bubbling aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22 - 22.6 °C

pH:

at t0 = 8.1 to 8.2
at t72h = 8.2 to 9.0

Nominal and measured concentrations:

Nominal concentrations: 0, 0.013, 0.024, 0.044, 0.084 and 0.16 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask
- Material, size, headspace, fill volume: flask of 30 mL with 20 mL of the test solution
- Aeration: none
- Type of flow-trough (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: around 10^4/mL
- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): sextuplicate

GROWTH MEDIUM
- Standard medium used: yes (from OECD guideline 201)

OTHER TEST CONDITIONS
- Photoperiod: constante (24h/24h)
- Light intensity and quality: day light (400-700 nm), around 7500 lux (equivalent to 55 µE.m-².s-1)


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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: between 1.8 and 2
- Test concentrations: 0, 0.013, 0.024, 0.044, 0.084 and 0.16

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

0.098 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CI 0.094-0.10)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: ErC20

Effect conc.:

0.057 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CI 0.053-0.061)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

0.041 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CI 0.037-0.045)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- ErC50 72h: 1.17 mg/L
- Results with reference substance valid? Yes. The ErC50 72h values falls within the acceptable range of 0.92 mg/L and 1.46 mg/L (as defined by the NF EN ISO 8692-2005 )
- Other: reference substance: Potassium dichromate

Reported statistics and error estimates:

- CErx% 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

Concentration mg/L	Cells concentration x 10^6
	1	2	3	4	5	6	Average cells number	Cells inhibition
0	4.34	5.68	5.18	5.98	4.16	5.32	5.11
0.013	5.74	5.96	6.12				5.94	-16%
0.024	5.44	5.36	5.68				5.49	-8%
0.044	2.26	2.46	2.28				2.33	54%
0.084	0.50	0.58	0.33				0.47	91%
0.16	0.04	0.04	0.04				0.04	99%
	Growth Rate
	1	2	3	4	5	6	Growth rate inhibition	Inhibition
0	2.02	2.11	2.08	2.13	2.01	2.09	2.08
0.013	2.12	2.13	2.14				2.13	-3%
0.024	2.10	2.09	2.11				2.10	-1%
0.044	1.81	1.84	1.81				1.82	12%
0.084	1.30	1.35	1.17				1.27	39%
0.16	0.43	0.46	0.49				0.46	78%

Validation criteria:

* Average growth rate for the control at 72 hours : 2.08 j-1 (> 0.92 j-1)

* Coefficient of variation of average specific growth rates during the whole test period in replicates control cultures : 2.1% (< 7%)

* The pH of the control medium did not increase by more than 1.5 units during the 72h period. (+0.9 pH units)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions:
72h - ErC50 = 0.098 mg/L (0.094-0.10 mg/L)
72h - NOEC = 0.024 mg/L
Results are not considered valid as the light intensity of the test was equal to 55 µE/m²/s1, which is above the recommanded range of 60-120 µE/m²/s1 by the guideline. As coloured substances can absorb photosynthetically active light and hence limit growth of algal cultures, the test should have been performed at the maximum recommanded light intensity, i.e. 120 µE/m²/s1 in order to reduce this growth inhibition phenomena.

Therefore, these low experimental values are most probably due to a growth inhibition because of the inadequate light intensity instead of a such inherent toxicity. Or, the potential inherent toxicity of the test item must have been aggravated be the phenomena of growth inhibition.

Executive summary:

The acute algal toxicity test for the substance Sepisol Fast Violet 3B 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 OECD guideline 201 (2006) was followed.

The median effective concentrations (ErC50) 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: 0.16, 0.084, 0.044, 0.024 and 0.013 mg/L.

 

A solution was prepared by diluting 2 mg of test material in 1 liter of the assay medium. No solvents were used to facilitate the dispersion of the test item.

 

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.

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

 

Under the experimental conditions:

    * 72h - ErC10 = 0.041 mg/L (0.037-0.045 mg/L)

    * 72h - ErC20 = 0.057 mg/L (0.053-0.061 mg/L)

    * 72h - ErC50 = 0.098 mg/L (0.094-0.10 mg/L)

    * 72h NOEC = 0.024 mg/L

Results are not considered valid as the light intensity of the test was equal to 55 µE/m²/s1, which is above the recommanded range of 60-120 µE/m²/s1 by the guideline. As coloured substances can absorb photosynthetically active light and hence limit growth of algal cultures, the test should have been performed at the maximum recommanded light intensity, i.e. 120 µE/m²/s1 in order to reduce this growth inhibition phenomena.

Therefore, these low experimental values are most probably due to a growth inhibition because of the inadequate light intensity instead of a such inherent toxicity. Or, the potential inherent toxicity of the test item must have been aggravated be the phenomena of growth inhibition.