1,3,5-Naphthalenetrisulfonic acid, 7-[2-[2-[2-[5-cyano-4-methyl-2,6-bis[(4-sulfophenyl)amino]-3-pyridinyl]diazenyl]-4-(2-naphthalenyl)-5-thiazolyl]diazenyl]-, lithium sodium salt (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:

Study range finding tests 2005-10-18 to 2005-10-31. Definitive tests 2005-11-22 to 2005-11-25. Study report signed 2006-01-03.

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:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Method C3 of Commission Directive 92/69/EEC

Deviations:

yes

Remarks:

(modified to differentiate the indirect effect of light absorption by the coloured test substance from any direct toxicity, as described in RCC Project 460302)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-980-9

EC Name:

-

Cas Number:

1016986-95-0

Molecular formula:

Hill formula: C42 H24 Lim N9 Nan O15 S6 m+n=5 3=

IUPAC Name:

7-{2-[2-(2-{5-cyano-4-methyl-2,6-bis[(4-sulfophenyl)amino]pyridin-3-yl}diazen-1-yl)-4-(naphthalen-2-yl)-1,3-thiazol-5-yl]diazen-1-yl}naphthalene-1,3,5-trisulfonic acid trilithium hydride disodium hydride

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each test group from Experiment A (shaded test vessels) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken on each occasion and stored at approximately -20 degree C for further analysis if necessary.
The test samples were analysed following filtration through glass wool to remove algal cells

Test solutions

Vehicle:

no

Details on test solutions:

The test material was dissolved directly in culture medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Name: Scenedesmus subspicatus
- Strain: CCAP 276/20
- Source: Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP) Dunstaffnage Marine Laboratory and maintained in the Safepharm laboratory
- Method of cultivation: Periodic replenishment of culture medium

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 +/-1 degree C

pH:

7.2 - 7.8

Nominal and measured concentrations:

All test concentrations were corrected for active ingredient (a.i.) content. Based on the results of the range finding tests the definitive test was carried out with test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations ranging from 87% to 103% of nominal and so it was considered justifiable to estimate the EC50 values in terms of nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel:250ml glass conical flasks covered by glass petri dishes
- Material, size, headspace, fill volume: Flasks contained 100ml of algal suspension and relevant test material. For Experiment A (exposed test vessels) the glass petri dishes contained 80ml of culture medium. For Experiment B (shaded test vessels) the glass petri dishes contained 80ml of each test preparation. The depth of preparation (15mm) in the petri dishes was half the depth of the preparation in the conical flasks (30mm).
- Initial cells density: 6350 cells/ml (Experiment A) 6050 cells/ml (Experiment B)
- Control end cells density: 166250 (Experiment A) 221250 (Experiment B)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis purified deionised water (Elga Optima 15+)
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was measured daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination (7000 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter - Coulter Multisizer Particle Counter
- Observation intervals: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined.

TEST CONCENTRATIONS
- Range finding study was conducted with test concentrations of 0.10, 1.0, 10 and 100 mg ai/l
- Test concentrations: Based on the results of the range finding test the following test concentrations were assigned to the definitive test 1.0, 3.2, 10, 32 and 100 mg ai/l

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

(Exposed vessel - Experiment A)

Remarks on result:

other: 95% CL 2.0 - 3.7 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

(Exposed vessel - Experiment A)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

(Exposed vessel - Experiment A)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

(Shaded vessel - Experiment B)

Remarks on result:

other: 95% CL 3.6 - 5.5 mg/l

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

(Shaded vessel - Experiment B)

Remarks on result:

other: 95% CL 18 - 37 mg/l

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

(Shaded vessel - Experiment B)

Details on results:

- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities observed.
- Adherence to test vessels: Stability tests showed no evidence of adherence to the test vessels

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's Test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the area under the growth curve data for Experiment A (Exposed test vessels) and B (Shaded test vessels) at 72 hours for the control and all test concentrations 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:

Exposure of Scenedesmus subspicatus to the test material in Experiment A (Exposed vessels - shaded by excess control culture medium) gave an EbC50 (72h) value of 2.7 mg ai/l, 95% confidence limits 2.0 - 3.7 mg ai/l and an ErC50 (0-72h) value of 34 mg ai/l. The No observed Effect Concentration was 1.0 mg ai/l. In Experiment A inhibition of algal growth was due to a combination of both the toxic effects of the test material and reduction in light intensity.
Exposure of Scenedesmus subspicatus to the test material in Experiment B (Shaded vessels - shaded by solutions of test material) gave an EbC50 (72h) value of 4.4 mg ai/l, 95% confidence limits 3.6 - 5.5 mg ai/l and an ErC50 (0-72h) value of 26 mg ai/l, 95% confidence limits 18 - 37 mg ai/l. The No Observed Effect Concentration was 1.0 mg ai/l. In Experiment B inhibition of algal growth was due to a reduction in light intensity alone.
Given that the shaded test vessels showed greater inhibition of algal growth it is concluded that the effect of the test material on algal growth was probably due to a reduction in light intensity alone, not the intrinsic toxic properties of the test material and therefore should not be used as a basis for classification.

Executive summary:

Introduction

This study was performed to assess the effect of the test substance on the growth of green alga Scenedesmus subspicatus. The test was conducted following the method described in:

OECD Guidelined for Testing of Chemicals (1984) No. 201, 'Alga, Growth Inhibition Test' referenced as Method C3 of Commission Directive 92/69/EEC

and further refined for coloured 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 coloured test substance (RCC project 460302).

Results

The 72 hour EC50 based on biomass for the exposed test vessels was 2.7 mg ai/l. The 72 hour EC50 based on growth rate for the exposed test vessels was 34 mg ai/l.

The 72 hour EC50 based on biomass for the shaded test vessels was 4.4 mg ai/l. The 72 hour EC50 based on growth rate for the exposed test vessels was 26 mg ai/l.

Conclusion

In this study the shaded test vessels showed greater inhibition of algal growth therefore it is concluded that the effect of the test material on algal growth was probably due to a reduction in light intensity alone, not the intrinsic toxic properties of the test material and that the EC50 values should not be used as a basis for classification.