Reaction product of 3-[4-(2-aminoethylamino)-6-(4-sulfoanilino)-1,3,5-heterocycle-2-ylamino]benzensulfonic acid, ammonia water, sodium chloride and {mixture of [poly(1~4)chlorosulfonylphthalocyaninato-N29,N30,N31,N32]copper(II), [poly(1~3)chlorosulfonyl (tribenzo[b,g,l]pyrido[2,3-q]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)]copper(II),[poly(1~2)chlorosulfonyl(dibenzol[b,g(or b,l)dipyrido[2,3(or 3,2)-l,q (or g,q)]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)] copper(II) and [monochlorosulfonyl(benzo[b]tripyridol[2,3(or 3,2)-g,lq]-5,10,15,20- tetraazaporphyrinato- N21,N22,N23,N24)] copper(II)

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:

18 November 2008 to 21 November 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Due to the coloured nature of the test solutions prepared the study was conducted using a modified algal inhibition test method with increased light intensity and decreased test volume in order to minimise the effects of light adsorption by the test material at the wavelengths required for photosynthetic growth.
Further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to coloured test substances.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 August 2008; Date of signature: 04 March 2010

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification : H-CB sodium salt
Description : blue solid
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: Cell density approximately 10 3(cubed) cells/ml
- Sampling method: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Samples were taken from the control (replicates R1 - R6 pooled) and the 100 mg/l test group (replicates R1- R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately -20°C for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (50 mg) was dissolved in culture medium and the volume adjusted to 250 ml to give a 200 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (25 ml) of each of the stock solutions was separately mixed with algal suspension (25 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
- Controls: yes, The control group was maintained under identical conditions but not exposed to the test material.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Not stated.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
- Acclimation period: Not stated.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: Not stated.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Test conditions

Hardness:

Not stated.

Test temperature:

24 ± 1°C

pH:

7.2 - 7.3

Dissolved oxygen:

Not stated.

Salinity:

Not applicable as freshwater used.

Nominal and measured concentrations:

Nominal concentrations:
Range finding study: 0.10, 1.0, 10 and 100 mg/l
Main study: 100 mg/l
Measured concentrations: 89.6 and 92.3

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flask
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 25 ml of test preparation, 25 ml of algal suspension
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): not applicable as static test conditions
- Initial cells density: approximately 10 3 cells/ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 10000 lux provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rates: average specific growth rate; Percentage inhibition of growth rate; Percentage inhibition of yield; 0, 24, 48 and 72 hours
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]

TEST CONCENTRATIONS
- Range finding study: Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/I for a period of 72 hours.
- Test concentrations: "limit test" was conducted at a concentration of 100 mg/l
- Results used to determine the conditions for the definitive study: Based on the result of the range-finding test a "limit test" was conducted at a
concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities observed.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
ErC50 (0 - 72 h): 0.52 mg/I, 95% confidence limits 0.43 - 0.62 mg/I
EyC50 (0 - 72 h): 0.29 mg/I, 95% confidence limits 0.25 - 0.33 mg/I

Reported statistics and error estimates:

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1 attached below. The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/l. Based on this information a single test concentration of six replicates, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no effect on growth was observed.

Definitive Test

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3 (attached below). Daily specific growth rates for the control cultures are given in Table 4 (attached below). Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5 (attached below).

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 64 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours: 4.28 x 10³ cells per ml

Mean cell density of control at 72 hours: 2.72 x 10⁵ cells per ml The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Verification of test concentrations

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No. 440/2008 and further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to coloured test substances.

Methods. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a concentration of 100 mg/I (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. Due to the coloured nature of the test solutions prepared the study was conducted using a modified algal inhibition test method with increased light intensity and decreased test volume in order to minimise the effects of light adsorption by the test material at the wavelengths required for photosynthetic growth.

Results. Exposure of Desmodesmus subspicatus to the test material gave EC50 values of greater than 100 mg/I and correspondingly the No Observed Effect Concentration was 100 mg/I. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Conclusion. The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.