N,N-bis(9,9-dimethylfluoren-2-yl)-4-phenyl-9,9'-spirobi[fluorene]-4'-amine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 19, 2014 - March 24, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine

EC Number:

700-681-0

Cas Number:

1364603-07-5

Molecular formula:

C52H37N

IUPAC Name:

Biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine

Test material form:

solid

Specific details on test material used for the study:

Batch: OS11008922
Appearance: Pale yellow powder without visible impurities
Water solubility: 1.7e-12 mg/L
Released until: August 15, 2016
Storage: Tightly closed, dark at room temperature (15 to 25°C)

Sampling and analysis

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 (1.7 e-12 mg/L calculated Episuite), the compound cannot be detected with standard analytical methods.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium (reconstituted water and test item) was prepared freshly. 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

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna Straus
Origin: Daphnia magna Straus was originally obtained from IBACON GmbH (Roßdorf, Germany).
Culture conditions: The clone is bred in the laboratories of of the test facility
Parental daphnids are used for reproduction until they are about 6 weeks old. Thereafter, they are replaced by neonates.
Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2°C and a 16 hour light and 8 hour dark regime to ensure similar conditions as in the experiment. Offspring are removed from the vessels at least twice per week.
Feeding: The parental daphnids are fed ad libitum with unicellular green algae Desmodesmus subspicatus three times per week.
Age: Offspring less than 24 hours old were used for the study.
Acclimation period: same as test

Feeding during test: None

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg CaCO3/L

Test temperature:

20.4-21.0°C

pH:

7.74 - 7.78

Dissolved oxygen:

8.16 - 8.26

Conductivity:

710 µS/cm

Nominal and measured concentrations:

Nominal 100 mg/L

Details on test conditions:

EXPOSURE:
The study was performed in an air-conditioned room. For the study 20 mL glass test tubes containing at least 20 mL either reconstituted water (control group) or test medium (test item group) were used. Each test vessel contained five daphnids resulting in 4 mL medium per daphnid. They were not fed and the media were not aerated during the exposure.

The test was performed as a static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
100 mg/L: 20 daphnids

CONCENTRATION(S)
In a pre-test no immobilization was observed at a concentration of 100 mg/L under open static conditions. Therefore, the solution of a nominal test item concentration of 100 mg/L was tested in the present study.

For the control, reconstituted water (ELENDT M4 medium) was used.

VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.74 - 7.78
- Total hardness: 250 mg CaCO3/L
- O2-Concentration: 8.16 - 8.26 mg/L
- Conductivity: 710 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 48 h


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark
- Light intensity: The mean light intensities were 644 Lux and 615 Lux prior to and at the end of the exposure period, respectively.

References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990

Reference substance (positive control):

no

Remarks:

No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 > 1.7e-12 mg/L

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 > 1.7e-12 mg/L

Details on results:

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of revealed no aquatic toxicity in the test system.


The 48h EC50 exceeded the water solubility of 1.7e-12 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

Potassium dichromate:
24h EC50: 1.221 mg/L (1.050 – 1.524 mg/L)
48h EC50: 0.819 mg/L (0.721 – 0.931 mg/L)

The positive control (Potassium dichromate) showed a 24h EC50 value of 1.221 mg/L which is within the range of the published data of 0.6 to 1.7 mg/L (Council Regulation (EC) No. 440/2008) and 0.6 to 2.1 mg/L (OECD Guideline No. 202).

Applicant's summary and conclusion

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 aquatic toxicity in the test system.
The 48h EC50 was >1.7e-12 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Executive summary:

Objective

The objective of this study was to evaluate the influence of the test item on the immobilization of Daphnia magna.

 

Study Design

For this purpose, juvenile daphnids were exposed to a nominal test item concentration of 100 mg/L (limit test) over 48 hours, in an open static system. The daphnids were observed for immobilization 24 and 48 hours after placing in the aqueous test item solution. The study comprised of one test item group with four test vessels containing five daphnids each, i.e. 20 daphnids in total. Additionally, one control group (20 daphnids) was used.

 

Results

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 (1.7e-12 mg/L), the compound cannot be detected with standard analytical methods and 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.

After exposure of daphnids to a nominal concentration of 100 mg/L for 48 hours, the following results were obtained:

 

Daphnids exposed to an aqueous preparation of a nominal concentration of 100 mg/L of  the test item were not affected.

 

EC50 values:

The following EC50 values for daphnids were determined:

24h EC50:       >1.7e-12 mg/L (nominal >100 mg/L)

48h EC50:       >1.7e-12 mg/L (nominal >100 mg/L)

 

Conclusions

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no aquatic toxicity in the test system.

The 48h EC50 was >1.7e-12 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.