5,5'-{ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-butyl-1H-pyrazole-4,1-diyl)]}diisophthalic acid

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:

The experimental start and completion dates were 24 and 28 May 2011. The report was issued on 04 August 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

modified to differentiate the indirect effect of light absorption, as described in RCC Project 460302

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

EC Number:

700-616-6

Cas Number:

849608-59-9

Molecular formula:

C36H32K4N14O8S4

IUPAC Name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.1, 3.7, 12, 38 and 120 mg/L
- Sampling method: 0h samples for analysis taken from excess test solutions. 72h samples from a single test replicate solution of each exposure (containing alga). All samples at 72 hours were analysed post centrifugation.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: Test medium
- Test solutions: nominal concentrations of 1.1, 3.7, 12, 38 and 120 mg/L.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: laboratory cultures at Brixham Environmental Laboratory
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Grown in test media

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

The hourly temperature measurements remained within 24 ± 2°C

pH:

At the start of the test the pH ranged from 7.3 to 7.4 and at the end of the test ranged from 6.7 to 6.8

Nominal and measured concentrations:

Test concentrations were culture medium control and test substance at nominal concentrations of 1.1, 3.7, 12, 38 and 120 mg/L. The overall geometric mean measured concentrations ranged from 78 to 119% of the nominal values. The concentrations of test substance were not maintained within ± 20 % of the nominal concentrations throughout the test, therefore the calculation and reporting of results is based on the geometric mean measured concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of 400 and 250 ml nominal capacity
- Type: closed
- Material, size, headspace, fill volume: 100ml test solution
- Initial cells density: 10500 cells/ml
- Control end cells density: 314000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Light intensity and quality: 10078 lux 'cool white' illunmination

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Test concentrations: 1.1, 3.7, 12, 38 and 120 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Exposed vessel

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 123 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Exposed vessels

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Exposed vessel

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Exposed vessel

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Exposed vessel

Remarks on result:

other: 95% CL 5.2-15 mg/L

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Exposed vessel

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Shaded vessel

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 123 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Shaded vessel

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Shaded vessel

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Shaded vessel

Effect concentrations 11

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Shaded vessel

Remarks on result:

other: 95% CL 4.0 - 11 mg/L

Effect concentrations 12

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Shaded vessel

Details on results:

- Exponential growth in the control (for algal test): yes

- Colour differences: The growth rate inhibition was essentially the same for Exposed vessels (shaded by excess control culture medium, E) and the Shaded vessels (shaded by test substance solution, S). At the two lowest tested concentrations (0.86 and 3.3 mg/L) the S/E values were 0.42 and 0.89 but this is not considered to alter the conclusion. Therefore, at these concentrations it can be considered that the effects on growth rate can be attributed to the effects of light absorption (shading) by the coloured test substance. However, at 123 mg/L mean inhibition in the exposure vessels was considerably greater than mean inhibition in the shaded vessels indicating a toxic response.

Reported statistics and error estimates:

The data for cell counts obtained at 24, 48 and 72 hours was statistically analysed using a laboratory in-house software package, PCADA, where appropriate procedures were applied to test for significant differences (p <0.05) between the control and test concentrations. EC50 values and the associated 95% confidence intervals were determined using the US EPA program ICPIN (version 2, June 1993)

Any other information on results incl. tables

From the inhibition curves it was concluded that the inhibition curves are essentially the same (S/E ≥0.9) up to 45 mg/L. At the two lowest tested concentrations (0.86 and 3.3 mg/L) the S/E values were 0.42 and 0.89 but this is not considered to alter the conclusion. Therefore, at these concentrations it can be considered that the effects on growth rate can be attributed to the effects of light absorption (shading) by the coloured test substance. However, at 123 mg/L mean inhibition in the exposure vessels was considerably greater than mean inhibition in the shaded vessels indicating a toxic response.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

It is concluded that the effects on growth rate at concentrations of 100 mg/L or less of test material are due to the effects of light absorption. At 123 mg/L there is evidence that growth retardation is due to a toxic effect.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) adopted 23 March 2006 and 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, and that due to reduction in growth by light absorption of the coloured test substance.

Methods

Following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous solution of the test material for 72 hours, under constant illumination and stirred continuously via magnetic stirrer at a temperature of 24 ± 1 °C. The test was conducted using two experimental methods performed in parallel.

Experiment A (Exposure Vessels)

In Experiment A the algae were exposed to test material concentrations of 1.1, 3.7, 12, 38 and 120 mg active ingredient (ai/l). Glass petri dishes above the test vessels contained culture medium alone. Therefore, any inhibition of algal growth in these test vessels was due to a combination of both the toxic effects of the test material and reduction in light intensity.

Experiment B (Shaded Vessels)

In Experiment B the glass petri dishes above the test vessels contained test material solutions at concentrations of 1.1, 3.7, 12, 38 and 120mg active ingredient (ai/l). The test vessels contained algal cells in culture medium alone. Therefore any inhibition of algal growth was due to a reduction in light intensity alone.

Results

Exposure Vessels

Exposure of Scenedesmus subspicatus to the test material gave an EyC50 (72 h) value of 9.5 mg ai/l and an ErC50 (0-72 h) value of >123 mg ai/l. The No Observed Effect Concentration at 72 hours was 0.86 mg ai/l based on growth rate.

Shaded Vessels

A reduction in light intensity resulted in reduction of algal growth. The EC50 values were calculated based on the concentration of the test material in the glass petri dishes above the test cultures. The reduction in growth of Scenedesmus subspicatus exposed to light passed through the test material solutions gave an ECy50 (72 h) value of > 7.2 mg ai/l and an ErC50 (0 -72 h) value of >123 mg ai/l. The No Observed Effect Concentration was 0.86 mg ai/l.

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 material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material.