Copper phthalocyanine direct dye

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:

24/11/2008 to 15/01/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

No further information requried.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The test material concentration in the test samples was determined spectrophotometrically using an external standard.
Standard solutions of test material were prepared in water at a nominal concentration of 5.0 mg/l.

- Sampling method:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. 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 and the 100 mg/l test group at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis:
Room temperature in the dark
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

- Sample preparation for analytical method
A volume of test sample was diluted with water to give a final theoretical concentration.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- 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. This stock solution was mixed with algal suspension (250 ml) to give the required test concentration of 100 mg/l.

- Controls:
The control group was maintained under identical conditions as the test material but not exposed to the test material.

- Evidence of undissolved material:
The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Desmodesmus subspicatus
- Strain:
CCAP 276/20
- Source (laboratory, culture collection):
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Method of cultivation:
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
- Culturing media and conditions (same as test or not):
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCI2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

- Any deformed or abnormal cells observed: All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

nda

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test.

pH:

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.7 - 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
The pH values of the test cultures were observed to increase from pH 7.3 at 0 hours to pH 7.5-7.7 at 72 hours.

Dissolved oxygen:

nda

Salinity:

nda

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
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.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 ml glass conical flasks each containing 25 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were used for each control and test concentration. The test material was dissolved directly in culture medium.

- Initial cells density:
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10E+4 - 10E+5 cells/ml.

- Control end cells density:
Mean cell density of control at 0 hours: 5.32 x 10E+3 cells per ml
Mean cell density of control at 72 hours: 1.05 x 10E+5 cells per ml

- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 6 flasks
- No. of vessels per control (replicates): 6 flasks

GROWTH MEDIUM
Culture medium:
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCI2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

OTHER TEST CONDITIONS
- Sterile test conditions: nda
- Adjustment of pH:
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
- Photoperiod: Constant Illumination
- Light intensity and quality: Approximately 10000 lux) provided by warm white lighting (380 - 730 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations:
An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 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. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.

- 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):
The data show that the cell concentration of the control cultures increased by a factor of 20 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.

- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

- Any stimulation of growth found in any treatment: nda

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

Results with reference substance (positive control):

- Results with reference substance valid:
The results from the positive control with potassium dichromate were within the normal range for this reference material.

- EC50:
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:

ErC50 (0- 72 h): 0.52 mg/l, 95% confidence limits 0.43- 0.62 mg/l
EyC50 (0- 72 h): 0.29 mg/l, 95% confidence limits 0.25- 0.33 mg/l
EbC50 (0- 72 h): 0.30 mg/l, 95% confidence limits 0.26- 0.34 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral: 0.125 mg/l

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P>_0.05), between the control and 100 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/l.

Any other information on results incl. tables

From the data given it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP276/20) were not affected by the presence of the test material at a nominal test concentration of 100 mg/l over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

At both the start and end of the test the control cultures were observed to be clear colourless solutions whilst the 100 mg/l test cultures were observed to be deep blue solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in test medium. A method of analysis was validated and proven to be suitable for use.

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:

In an algal growth inhibition test on the growth of Desmodesmus subspicatus (Harlan project number: 0959/0232) the test material was found to have EC₅₀ values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

The method followed the recommendations of 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.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/I.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 97% to 111% of nominal and so the results are based on nominal test concentrations only.