Reaction product of {Sulfonic acid derivatives of [4-(aminoalkylamino)-6-substituted-1,3,5-triazin-2-ylamino]benzene}, ammonia water, sodium chloride and [poly(1~4)chlorosulfonyl(poly(1~4) benzo-poly(3~0)pyrido-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:

Experimental starting date: 1 November 2013; Experimental completion date: 21 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to colored test substances.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to colored test substances.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: CIM-25
Description: dark purple powder
Sponsors description: dark blue powder
Batch: MB-1
Expiry Date: 31 March 2014
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/L

- Sampling method:
Samples were taken from the control and the 100 mg/L test group (replicates R1 – R6 pooled) at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis:
AlI 0-Hour samples were stored frozen prior to analysis.
Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

RANGE-FINDING TESTS:
The test concentration to be used in the definitive test was determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.7 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

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

The control group was maintained under identical conditions but not exposed to the test item.

Given that the test material formed colored test solutions it was considered appropriate to conduct spectrophotometer measurements on the stock solutions in order to determine whether significant adsorption of light at the wavelengths required for photosynthesis occurred (460 and 665 nm).

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 -730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

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

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

The results of the initial range-finding test showed that significant inhibition of growth occurred at both 10 and 100 mg/L where significant light absorbance at 665 nm had been observed. It was therefore considered appropriate to conduct a second range-finding test following the modifications recommended in the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to colored test substances.

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. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

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

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the second range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

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

DEFINITIVE TEST:
Based on the result of the second range-finding test a "limit test" was conducted at a concentration of 100 mg/L under modified test conditions to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.

Experimental Preparation:
An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (4.8 mL) to give the required test concentration of 100 mg/L.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test Species:
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°C.

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 10E3 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 10E4 – 10E5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable.

Test conditions

Test temperature:

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

pH:

The culture medium pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure.

Nominal and measured concentrations:

Definitive study:
Nominal: 100 mg/L
Measured: 106% to 112% of nominal.

Details on test conditions:

DEFINITIVE TEST SYSTEM/EXPOSURE CONDITIONS:
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 25 mL of test preparation were used for the control and 100 mg/L treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.16 x 10E5 cells per mL. Inoculation of 500 mL of test medium with 4.8 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

GROWTH MEDIUM
- Standard medium used: yes
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growh rate and yield

Details on results:

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

Water Quality Data:
The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 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.

Observations on Test Item Solubility:
At the start of the test all control cultures were observed to be clear colorless solution whilst the 100 mg/L test cultures were observed to be dark blue solutions. After the 72-Hour test period the control cultures were observed to be green dispersions whilst the 100 mg/L test cultures were observed to be dark blue solutions.

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): 1.2 mg/L; 95% confidence limits 1.0 - 1.3 mg/L
EyC50 (0 - 72 h): 0.52 mg/L; 95% confidence limits 0.45 - 0.62 mg/L

NOEC based on growth rate: 0.25 mg/L
NOEC based on yield: 0.25 mg/L
LOEC based on growth rate: 0.5 mg/L
LOEC based on yield: 0.5 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

Results

Range-finding Tests:

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding tests are given in Table 1 and Table 2 (see attached background material).

The results of the initial range-finding test performed using standard conditions showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100mg/L. The results of the second range-finding test showed no effect on growth at any of the test concentrations employed.

During the initial range-finding test, spectrophotometer measurements taken at the wavelengths required for photosynthetic growth (460 and 665 nm) showed that the most significant adsorption occurred at 665 nm at both 10 and 100mg/L (see Table 3 – attached background material). The use of a modified algal inhibition test with increased light intensity and decreased test sample volume overcame this adsorption effect resulting in no significant effect on the algal cells present.

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 116% of nominal.

Based on this information a single test concentration of six replicates, of 100 mg/L under modified test conditions 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

Verification of Test Concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 106% to 112% of nominal and so the results are based on nominal test concentrations only.

Growth Data:

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 4. Daily specific growth rates for the control cultures are given in Table 5. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 6.

See attached background material for Tables 4-6.

From the data given in Table 4 and Table 6, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata(CCAP 278/4) were not affected by the presence of the test item 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.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate:

ErC₁₀ (0 - 72 h): >100 mg/L

ErC₂₀ (0 - 72 h): >100 mg/L

ErC₅₀ (0 - 72 h): >100 mg/L

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.

Inhibition of Yield:

EyC₁₀ (0 - 72 h): >100 mg/L

EyC₂₀ (0 - 72 h): >100 mg/L

EyC₅₀ (0 - 72 h): >100 mg/L

Statistical analysis of the yield data was carried out as for growth rate. There were no statistically significant decreases in yield (P≥0.05), between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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 761/2009 and further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to colored test substances.

Methods

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Due to the colored 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 minimize the effects of light adsorption by the test item at the wavelengths required for photosynthetic growth.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 106% to 112% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀ values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

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