Dodeca (lithium, sodium)-2-({4-[4-(4-{bis[alkyl-(sulfonatoalkoxy)-4-({-sulfonato-[(substituted-phenyl)diazenyl]phenyl} diazenyl)anilino]-triazin-yl}-6-[alkyl-(sulfonatoalkoxy)-4-(sulfonato--[(substituted-phenyl)diazenyl]phenyl)diazenyl)anilino]piperazin-yl)-triazin-ylamino]-alkyl-5-(sulfonatoalkoxy)phenyl}diazenyl)-5-[(sulfonatophenyl)diazenyl]benzensulfonate

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:

26-29 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted following the GLP principles, and according to the OECD and Japanese guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute test" Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, November 21, 2003; the latest revision November 20, 2006

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): E-BW102
- Substance type: reddish brown powder
- Physical state: solid
- Lot/batch No.: MB-1
- Storage condition of test material: room temperature, shaded, air-tight

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0; 10; 18; 32; 56 and 100
- Sampling method: 1.0 ml from the middle layer of all vessels and mix
- Sample storage conditions before analysis: not reported

Test solutions

Vehicle:

no

Details on test solutions:

The stock solution was prepapred by addition of 100 mg test substance to 1000 ml dilution water, resulting in a concentration of 100 mg/L. From this stock solution, 75 ml were taken and added in a 300 ml Erlenmeyer flask with air-permeable silicon cap. this was considered as the highest test concentration of 100 mg/L. Next, by diluting the stock solution, series dilutions in 75 ml medium separated by a factor of 1.78 were prepared.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection


ACCLIMATION
- Acclimation period: 20-26 October 2010
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Study design

Test type:

other: static and shaking (100 rpm)

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

50 mg NAHCO3/L

Test temperature:

21.7 - 22.5

pH:

7.8 - 8.0

Nominal and measured concentrations:

Nominal: control, 10, 18, 32, 56 and 100 mg/L
Measured (TWM): 9.81; 17.7, 32.5, 51.8 and 99.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): open; air permeable cap
- Material, size, headspace, fill volume: glass, 300 ml
- Initial cells density: 5 x 10^3 cells/ml algae of exponential growth phase
- Control end cells density: 161 X 10^4 cells/ml (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes, Gorham


OTHER TEST CONDITIONS
- Sterile test conditions: yes, every 6 months
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 110 to 120 uE/m2/s, fluorescent white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 50% growth inhibition concentration and no observed effect concentration (0-72 h)
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: for the final test only 0; 10; 18; 32; 56 and 100 mg/L. The test solutions were colorless in control and brown in the rest of concentrations
- Range finding study: yes
- Test concentrations:control; 0.10; 1.0; 10 mg/L
- Results used to determine the conditions for the definitive study: 47.9 % inhibition was observed at 100 mg/L in the range finding test. As the test substance was of browncolor, additional studies were conducted on effects of light absorption or interception caused by the substance on algal growth. the methods used were as follows:
1) Growth of algae under the light filtered through petri dishes filled with the 100 mg/L test solution (concentration group) was measured to investigate the effects the effect of light absorption or interception by test solutions and test cultures. Light filtered through petri dish filled with test medium was used as control.
2) The effect of light absorption or interception by test solution and test cultures was investigated by reducing the test solution volume, thus reducing the light path. One replicate 300 ml Erlenmeyer falsks were each filled with 50; 75 or 100 ml of test solutions at EC10 (10 mg/L) or EC50 (100 mg/L).

both experiments were conducted under the same conditions as the final test.

The results of the experiment 1) indicate 37 % growth inhibition in the 100 mg/L group compared with the control group. the light intensity at the bottom of the concentration group was 12 uE/m^2/s as compared with 67 uE/m^2/s of the control group.

Regarding experiment 2), in the Erlenmeyer flask filled with 50 ml test medium and 100 mg/L, a 23.7% growth inhibition was measured and the light intensity at the bottom of the flask was 22 uE/m^2/s. The results of test conducted 100 ml test medium and 100 mg/L test substance indicated 36.4 % growth inhibition and a measured the light intensity at bottom of the flask of 13 uE/m^2/s. In case of the experiments conducted with 10 mg/L test substance in 50 ml and 100 ml media, the results indicated a percentage inhibition of -1.6 and 2.9 with the measured light intensities at the bottom of 61 uE/m2/s and 49 uE/m2/s, respectively.

The above results indicate a limitation of photosynthetic activity of the algae caused by the absorption or interception of lightin the test solutions colored by the test substance.

The final test was conducted in 75 ml and light intensity of 110-120 uE/m2/s in order to reduce the growth inhibition caused by the coloration of test solutions.

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.

Remarks:

the measured concentration was within 20% of nominal

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.81 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

NOECr was determined by ANOVA, Williams test, subsequent to Barlett test for homogeneity of variances

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- EC50: 0.698 mg/L (95% C.I. 0.660-0.739 mg/L), exposure period 8-11 Jun 2010
- Other: the average values of ErC50 since June 2000 were 0.815 +/- 0.085 mg/L, n=21
Min-max = 0.687 - 0.965 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance was a water-soluble and colored therefore causing the coloration of the test solutions. Growth inhibition resulting from the attenuation of light essential for the algal growth in the test solution was observed. The EC50 was determined to be > 100 mg/L.