Cuprate(4-), [[3,3',3'',3'''-[(29H,31H-phthalocyanine-1,8,15,22-tetrayl-.kappa.N29,.kappa.N30,.kappa.N31,.kappa.N32)tetrakis(sulfonyl)]tetrakis[1-propanesulfonato]](6-)]-, sodium (1:4), (SP-4-1)-

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:

2013-12-03 to 2013-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 1,8,15,22-tetra-(3-sulfoxypropylsulfonyl)copper phthalocyanine

EC Number:

696-145-8

Cas Number:

944730-39-6

Molecular formula:

C44H36CuN8O20S8 .4Na

IUPAC Name:

tetrasodium 1,8,15,22-tetra-(3-sulfoxypropylsulfonyl)copper phthalocyanine

Details on test material:

- Physical state: solid
- Purity: 96.9% w/w
- Lot/batch No.: RD3836 Batch 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from Experiment A only from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
Analysis of the test preparations from Experiment B was not performed as this was not a requirement of the test guidelines.
- Sample storage conditions before analysis: The 72-Hour test samples were analyzed on the day of receipt, the 0-Hour test samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

For the purpose of the definitive test, the test item was dissolved directly in culture medium using two parallel experimental set ups.

Experiment A – Experimental Preparation
Amounts of test item (120 and 38 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 120 and 38 mg/L stock solutions respectively. A series of dilutions were made from these stock solutions to give further stock solutions of 12, 3.8 and 1.2 mg/L. An aliquot (900 mL) of each of these stock solutions was separately inoculated with algal suspension (8.9 mL) to give the required test concentrations of 1.2, 3.8, 12, 38 and 120 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Experiment B – Experimental Preparation
Amounts of test material (120 and 38 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 120 and 38 mg/L stock solutions respectively. A series of dilutions were made from these stock solutions to give further stock solutions of 12, 3.8 and 1.2 mg/L. An aliquot (80 mL) of each of the 1.2, 3.8, 12, 38 and 120 mg/L stock solutions was separately placed in a glass petri dish, the overall depth of which was approximately half the depth of the test solutions in the conical flasks below.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Observations on Test Item Solubility
Experiment A
At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from light blue solutions at 1.2 mg/L through to dark blue solutions at 120 mg/L. After the 72-Hour test period the control cultures were observed to be pale green dispersions. The test cultures were observed to range from light blue/green dispersions at 1.2 mg/L through to dark blue solutions at 120 mg/L.

Experiment B
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.2 and 3.8 mg/L test cultures were observed to be pale green dispersions. The 12 mg/L test cultures were observed to be very pale green dispersions whilst the 38 and 120 mg/L test cultures were observed to be clear colorless solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. 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 ºC.

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 103 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 ºC until the algal cell density was approximately 104 - 105 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test conditions

Test temperature:

24 +/- 1ºC

pH:

7.2-7.6

Details on test conditions:

Experiment A – Experimental Preparation
In order to determine the inhibition of algal growth due to a combination of the toxic effects of the test item and the reduction in light intensity, algae were exposed to the test item at concentrations of 1.2, 3.8, 12, 38 and 120 mg/L, with glass petri dishes containing culture medium alone being placed above the test vessels.

Experiment B – Experimental Preparation
In order to determine the inhibition of algal growth due to light intensity alone, the test vessels contained algal cells in culture medium alone, whilst separate glass petri dishes containing the test item solutions at concentrations of 1.2, 3.8, 12, 38 and 120 mg/L were placed above the test vessels.

250 mL glass conical flasks were used.

For Experiment A six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. Glass petri dishes containing 80 mL of culture medium were placed above each of the conical flasks.

For Experiment B three flasks each containing 100 mL were used for each treatment group. Glass petri dishes containing 80 mL of each test preparation were placed above each of the conical flasks. The control data obtained for Experiment A was shared with Experiment B. The control was maintained under identical conditions but not exposed to the test item.

The depth of preparation (15 mm) in the petri dishes for both Experiment A and B was half the depth of the preparation in the conical flasks (30 mm).

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.06 x 105 cells per mL. Inoculation of an appropriate volume of test medium with this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were incubated (ETAD incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) and constantly stirred at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 8.4 - 11 mg/L Experiment A

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Experiment A

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Experiment A

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Experiment B

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Experiment B

Effect concentrations 12

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Experiment B

Details on results:

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 25 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 : 5.36 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.32 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Observations on Cultures
All test and control cultures for Experiment A and B were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Analysis of the test preparations taken from Experiment A at 0 and 72 hours showed measured test concentrations to range from 99% to 110% of nominal and so the results are based on nominal test concentrations only.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test item on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test item. Therefore, for classification purposes the results determined from Experiment A should be used.

Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. The method followed was designed to be compatible with theOECD 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. 761/2009, and further refined for colored test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the colored test substance (Memmertet al1994).

Results

Exposure of Pseudokirchneriella subcapitatato the test item in Experiment A gave the following results:

 

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	30		*		3.8	12
Yield	9.6	8.4	-	11	3.8	12

 

These results indicate the combined toxic nature of the test item and the reduction in light intensity.

 

In Experiment B, reduction in light intensity resulted in reduction of algal growth. The following results were calculated based on the concentration of the test item in the glass petri dishes above the test cultures:

 

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	75		^*		3.8	12
Yield	10		^*		3.8	12

 

*It was not possible to calculate 95% confidence limits for these EC₅₀values as the data generated did not fit the models available for the calculation of confidence limits.

Conclusion

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test item on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic propertiesof the test item. Therefore, for classification purposes the results determined from Experiment A should be used.