Mixture of hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-5-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate and hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-7-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate

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

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): E-BK105
- Substance type: black 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; 1.0; 3.2; 10; 32 and 100
- Sampling method: 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 etst concentration of 100 mg/L. Next, by diluting the stock solution, series dilutions in 75 ml medium separared by a factor of 3.16, were prepared.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 at 100 rpm

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

50 mg NAHCO3/L

Test temperature:

22.0 - 22.4

pH:

7.8 - 8.5

Nominal and measured concentrations:

Nominal: 0; 1.0; 3.2; 10; 32 and 100 mg/L
Measured: 0.95; 3.14; 9.86; 31.4; 99.2 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; 1.0; 3.2; 10; 32 and 100 mg/L. The test solutions were colorless in control, dark blue in 1.0 and 3.2 mg/L and black in 10; 32 and 100 mg/L.
- Range finding study: yes
- Test concentrations:control; 0.10; 1.0; 10 mg/L
- Results used to determine the conditions for the definitive study: 46 % inhibition was observed at 10 mg/L in the range finding test. As the test substance was of black color, 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 10 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 EC50 (10 mg/L) or EC100 (100 mg/L). In the Erlenmeyer flask filled with 50 ml test medium and 100 mg/L, it was noticed a 78.7 growth inhibition and the light intensity at the bottom of the flask was 5 uE/m^2/s. The results of test conducted 100 ml test medium and 100 mg/L test substance indicated 90.0 growth inhibition and a measured the light intensity at bottom of the flask of 3 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 indicates a percentage inhibition of 10.6 and 21 with the measured light intensities at the bottom of 40 uE/m2/s and 20 uE/m2/s, respectively.
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.

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

The results of the experiment 1) indicate 38.5 % growth inhibition in the 10 mg/L group compared with the control group. the light intensity at the bottom of the concentration group was lower as comparedto that of the control.

From the experiment 2) a trend was observed that the reduction of test solution volume increased the light intensities at the bottom of flsk in both 10 and 100 mg/L concentration groups, which in turn reduced the inhibitory effect.
Growth inhibition was attibuted to the inhibition of photosynthesis and not to the effects of the substance on algae.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.4 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

determined by least squared linear regression analysis

Basis for effect:

growth rate

Remarks on result:

other: 20.7-22.2

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.14 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal was 3.2 mg/L

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 21.4 mg/L.