14,14',15,15'-tetradehydro-7,7'-spirobi[dibenzo[b,g][1,9,4,6]dioxadiazacyclododecine]

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:

08/03/2011-11/03/2011

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:

2011

Report date:

2011

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 toxicity 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

Reference substance name:

14,14',15,15'-tetradehydro-7,7'-spirobi[dibenzo[b,g][1,9,4,6]dioxadiazacyclododecine]

EC Number:

700-609-8

Cas Number:

1231148-36-9

Molecular formula:

C31H24N4O4

IUPAC Name:

14,14',15,15'-tetradehydro-7,7'-spirobi[dibenzo[b,g][1,9,4,6]dioxadiazacyclododecine]

Constituent 2

Reference substance name:

SX-1

IUPAC Name:

SX-1

Details on test material:

- Name of test material (as cited in study report): SX-1
- Substance type: yellow-white powder
- Physical state: solid
- Analytical purity: 99.8%
- Lot/batch No.: 008809
- Expiration date of the lot/batch: not reported
- Stability under test conditions: stable
- Storage condition of test material: room temperature, shaded

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.14 mg/L
- Sampling method: (at the beginning of exposure) 1ml of test solution from the middle layer of all the test vessels and mix (diluted suitably with purified water if it was predicted that the concentrations will exceed the ones of the calibration curve). Next, 0.75 ml of solution and 0.75 ml of acetonitrile were mixed and the resulting solution was analysed by LC/MS.
(at the 24, 48, 72 hof exposure): 1ml of test solution from the middle layer of all the test vessels and mix. next, the samples were centrifugated for 10 min at 3000 rpm followed by collection of 0.75 ml supernatant diluted suitably with purified water if it was predicted that the concentrations will exceed the ones of the calibration curve). Next, 0.75 ml supernatant and 0.75 ml acetonitrile e were mixed and the resulting solutions were analysed by LC/MS.
- Sample storage conditions before analysis: not indicated

Test solutions

Vehicle:

yes

Details on test solutions:

The test solutions were prepared as follows:

Preparation of stock solution I: 28 mg of test substance were dissolved in 20 ml N,N-dimethylformamide and sonicated for 1 min. The resulting concentration was of 1400 mg/L .

Preparation of stock solution II: 100 ul of stock solution I were mixed with 1000 ml of medium at 22 deg C. The stcok solution was added to the medium while stirring, and then the resulting solution was sonicated for 30 min. The resulting concentration of stock solution II was 0.14 mg/L.

Preparation of the solvent stock solution: 100 ul dimethylformamide was mixed with the 1000 ml of medium at 22 deg C. The solvent was added to the medium while stirring, and then the solution was sonicated for 30 min.

The final preparation of the test solution was in in 300 ml Erlenmeyer flasks with air-permeable silicon cap. the final volume of each solution was 100 ml and the solvent concentration was 100 ul/L. There were 6 vessels/test group and 1 extra vessel was included in each test group.
The test group were as follows:
- control - just media, no stock solution II and not solvent stock solution
- solvent control - 100 ml solvent stock solutiom
- 0.14 mg/L - 100 ml of stock solution II

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): Preculture period from March 4 to 8, 2011
- Method of cultivation: The algae have been maintained by period subculture using Gorham medium. The algae were kep under sterile conditions and sterile test were [performed every 6 months in order to confirm that the algae were not contaminated

ACCLIMATION
- Acclimation period: 4-8 March 2011
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

In all test groups, color of test cultures were observed with the naked eye. A tendency of the medium to get more greenish during the exposure period was observed.
By microscopic observation at the end of the exposure, in concentration group 1, neither unusual cell shape of algae (contraction, expansion, damaged cell, etc.) not agglutionation was observed, and the algae looked normal compared with that in the control and solvent control groups.

Test conditions

Hardness:

50 mg/L NaHCO3

Test temperature:

21.5 - 21.7 deg C

pH:

7.9 - 8.4

Nominal and measured concentrations:

nominal: 0.14 mg/L
mean measured concentration: 0.113 mg/L (time weighted average)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer
- Type (delete if not applicable): open (air-permeable)
- Material, size, headspace, fill volume: glass, 300 ml, fill volume 100 ml/vessel
- Aeration: no
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 189 X 10^4 cells/ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 65-75 uE/m^2/s


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 50% growth inhibition concentration and no observed effect concentration (NOEC) (0-72 h)
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] electronic particle counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Only 0.14 mg/L was used
- Justification for using less concentrations than requested by guideline: In the range finding test, solvent control, 0.0014; 0.014, and 0.14 mg/L were tested. No inhibition of the algae was observed at the any of the concentrations from the range finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.113 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.113 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: the decrease in the concentration of the test substance was due to the adsorption of the test substance to the algae
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: adsorption to the algae
- Effect concentrations exceeding solubility of substance in test medium: no effects observed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.835 mg/L (95% C.L. 0.809-861 mg/L)
- Other: Growth inhibition of the reference substance was conducted periodically, every 6 months.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the current test, SX-1 was not found to be toxic to the green algae. Owing to the low water solubility of the test substance (<0.1 mg/L) no effects were observed up to the limit of solubility (EC 50 > 0.113 mg/L and NOEC >= 0.113 mg/L). Based on the current results and owing to the low water solubility (<0.1 mg/L), SX-1 should not be classified as Acute 1.