Coupling reaction products of 3,3’-dichlorobiphenyl-4,4’-bis(diazonium) dichloride with N-(2,4-dialkylphenyl)-3-oxobutanamide and potassium 4-[(3-oxobutanoyl)amino]substituted benzene and their basic aluminium and earth alkali metal salts

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:

28 December 2007 - 6 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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: Algal Growth Inhibition Test stipulated in the "Testing Methods for New Chemical Items" (November 21, 2003; No. 1121002, MHLW; November 13, 2003, No. 2, METI; No. 031121002, MOE, Japan, Partial amendment November 20, 2006)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): S-500
- Substance type: yellow powder
- Physical state: solid
- Lot/batch No.: MF1456-2345
- Stability under test conditions: stable (confirmed by IR)
- Storage condition of test material: dark storage place at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L (nominal) and control
- Sampling method: At the start, the solution sampled from the container for preparation in each test level was used for the measurement. At the end of the exposure, equal volume of the test solution was taken out from the test vessels in each test level and mixed, and then the solution after removal of algae by centrifugation (10 min, 3000 rpm) was used for the measurement.
- Sample storage conditions before analysis: not applicable.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: mixing and stirring by magnetic stirrer for 48 h, followed by suction filtration with 0.4 µm glass fibre filter and 0.2 µm membrane filter.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 mg/l (nominal)

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: old name: Selenastrum capricornutum
- Strain: ATCC 22662
- Source (laboratory, culture collection): originally from American Type Culture Collection (in 1995) and cultured in the laboratory.
- Age of inoculum (at test initiation): 3 days

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not indicated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

not indicated

Test temperature:

23.0 - 23.2 °C

pH:

7.9 - 8.0

Dissolved oxygen:

not measured

Salinity:

not applicable

Nominal and measured concentrations:

0 and 100 mg/l (nominal)
Measured concentrations: not determined, i.e. < 0.0069 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: sterilized 500 mL erlenmeyer flask with gas-permeable silicon rubber plug. Fill volume: 100 mL
- Aeration: no, but continuous shaking (approx. 100 rpm)
- Initial cells density: 10000 cells/mL
- Control end cells density: 630000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 90-95 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 0, 24, 48 and 72 h
- Determination of cell concentrations: particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study:
- Test concentrations: 10%, 32% and 100% of a 100 mg/L nominal stock solution.
- Results used to determine the conditions for the definitive study: no cell growth inhibition observed at any test concentration.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Effect concentration > solubility in medium

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities in control and exposed cells.
- Any stimulation of growth found in any treatment: mean cell concentration in exposed cells after 72 h: 790000 cells/mL (SD=34000). Mean cell concentration in control cells after 72 h: 630000 cells/ml (SD=64000).
- Effect concentrations exceeding solubility of substance in test medium: no observed effects at levels above the solubility of the substance in test medium.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.73 mg/L (growth rate)

Reported statistics and error estimates:

F test: to determine the homogeneity of variance for the data
Aspin-Welch t-test: to estimate the significant difference in comparison with control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted as a limit test in order to confirm the effect on the test organisms at around the solubility of the test item in medium. As a result, the concentrations of the test item in the test solutions during the exposure were below the determination limit (<0.0069 mg/L), indicating that the concentrations of the dissolved test item in medium was not clarified. However, no adverse effect was found in the definitive study. Therefore, it was decided that the test item had no adverse effect on the test organisms at around the solubility in medium. The environmental conditions were within the suitable range, therefore, it is concluded that this study complied with the applied test guidelines.