lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)

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:

2014-02-07 to 2014-02-25

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:

lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)

EC Number:

940-433-0

Molecular formula:

C23H14Li2N4O9S4

IUPAC Name:

lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 120 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis.

Test solutions

Details on test solutions:

A nominal amount of test item (60 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 120 mg/L stock solution. The pH of this stock solution was determined and adjusted to pH 7.5 prior to inoculation with 5.8 mL of algal suspension to give the required test concentration of 120 mg/L.

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

At the start of the test all control cultures were observed to be clear colorless solutions whilst the 120 mg/L test cultures were observed to be dark orange/red colored solutions. After the 72-Hour test period all control cultures were observed to be pale green dispersions whilst the 120 mg/L test cultures were observed to be dark orange/red colored dispersions.

Green coloration of the test preparations occurs over the test period due to the growth of the algal cells present, the depth of coloration depending upon the increase in cell density.

Test organisms

Test organisms (species):

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

Details on test organisms:

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 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 C until the algal cell density was approximately 104 - 105 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 +/- 1ºC

pH:

7.5 to 7.6

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 103 to 108% of nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

250 mL glass conical flasks were used. Six flasks each containing 25 mL of test preparation were used for the control and 120 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 429000 cells per mL. Inoculation of 500 mL of test medium with 5.8 mL of this algal suspension gave an initial nominal cell density of 5000 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

EFFECT PARAMETERS MEASURED :
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:

EC10

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The following data show that the cell concentration of the control cultures increased by a factor of 169 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 : 5430 cells per mL 5
Mean cell density of control at 72 hours : 919000 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 17% 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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 the start of the test all control cultures were observed to be clear colorless solutions whilst the 120 mg/L test cultures were observed to be dark orange/red colored solutions. After the 72-Hour test period all control cultures were observed to be pale green dispersions whilst the 120 mg/L test cultures were observed to be dark orange/red colored dispersions.

Green coloration of the test preparations occurs over the test period due to the growth of the algal cells present, the depth of coloration depending upon the increase in cell density.

Results with reference substance (positive control):

The positive control was conducted between 21 October 2013 and 24 October 2013.

A positive control (Harlan Study Number 41303826) 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.

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 120 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).

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 120 mg/L. The No Observed Effect Concentration was 120 mg/L.

Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata. 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.

Results & Conclusions

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 120 mg/L. The No Observed Effect Concentration was 120 mg/L.