4-bromo-9,9'-Spirobi[9H-fluorene]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 28, 2014 - Nov 13, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

PREPARATION OF THE TEST MATERIAL
The test medium (reconstituted water and test item) was freshly prepared. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

Analytical monitoring:

no

Remarks:

The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (0.00022 mg/L) the compound cannot be detected with standard analytical methods.

Vehicle:

yes

Details on test solutions:

Vehicle:

Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60

FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100

H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700

NaHCO3 50.0

- pH: 8


Preparation of the Test Item:
The test medium (reconstituted water and test item) was freshly prepared. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Desmodesmus subspicatus
- Source (laboratory, culture collection): Pseudokirchneriella subcapitata, strain 86.81 SAG was obtained from „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany.
- Method of cultivation:
The algae were taken from an exponentially growing preculture (growth rate: 73.0) which was set up 3 days prior to the experimental part under the same conditions as in the final study.

ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

-

Hardness:

about 24 mg/L as CaCO3

Test temperature:

21.8-22.5°C

pH:

7.76 - 8.39

Nominal and measured concentrations:

Nominal concentrations: 100.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per ml
- Control end cells density: 430455 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The light intensity measured immediately before start of the study was 7144 Lux ± 4.1% and 7155 Lux ± 6.0% at the end of the study.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L

Reference substance (positive control):

no

Remarks:

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.00004322 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.00004322 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.00004322 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > 0.00004322 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > 0.00004322 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > 0.00004322 mg/L

Details on results:

No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

The EC values exceeded the water solubility of 0.00004322 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

Study design:
The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L were dissolved in 100 mL reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Results:
The test item Art. 104864 (potassium dichromate) showed 72h EC values which are within the recommended range (Biomass integral EC50 0.20 – 0.75 mg/L and Growth rate EC50 of 0.60 –1.03 mg/L)

Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.

The following EC values (Growth rate and Yield) were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	>0.00004322 mg/L (nominal >100 mg/L) n.d.	>0.00004322 mg/L (nominal >100 mg/L) n.d.
EC20 [mg/L] 95% confidence interval	>0.00004322 mg/L (nominal >100 mg/L) n.d.	>0.00004322 mg/L (nominal >100 mg/L) n.d.
EC50 [mg/L] 95% confidence interval	>0.00004322 mg/L (nominal >100 mg/L) n.d.	>0.00004322 mg/L (nominal >100 mg/L) n.d.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.
The 72h EC50 and 72h EC10 for growth rate were >0.00004322 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Executive summary:

In this study the influence of the test item on the growth and growth rate of the green algae speciesDesmodesmus subspicatuswas tested.

The study design included one control group and one test item group with six replicates, each containing 100 mL reconstituted water or test medium and about 10000 cells/mL at the start of the experimental phase.

The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L.

The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item group and control group.

The test item concentration in the reconstituted water was not quantified at the start and the end of this study due to the low water solubility (0.00004322 mg/L). Because of the low water solubility, the compound cannot be detected with standard analytical methods. The development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification.

An aqueous preparation of a nominal concentration of 100 mg/L of the test item revealed no toxic effect in Desmodesmus subspicatus. Therefore, the 72h EC50 and 72h EC10 for growth rate  were >0.00004322 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Description of key information

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201.Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.The 72h EC50 and 72h EC10 for growth rate were >0.00004322 mg/L(nominal >100 mg/L) and, thus, could not be determined in this study.