4-[trans-4-(trans-4-butylcyclohexyl)-cyclohexyl]-1-ethoxy-2, 3-difluorobenzene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 29, 2013 - September 12, 2013

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)

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 (<0.033 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: 7.86
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
The test medium (reconstituted water and test item) was freshly prepared. Therefore, the calibrated flask with test item and vehicle (reconstituted water) 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 filter membrane (pore size 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): The test system used for the present study was Desmodesmus subspicatus, strain 86.81 SAG. The strain was supplied by: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, 37073 Göttingen, Germany. This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with a water temperature of 21.0 – 24.0°C, controlled at
± 2°C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany) and lit between 4440 and 8880 Lux. Fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.


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:

24 mg/L as CaCO3 (calculated)

Test temperature:

21.7 - 23.0°C

pH:

7.78 - 8.48

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: 564 750 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 7404 Lux ± 3.8% and 7219 Lux ± 2.8% at the end of the study. Thus, the light intensity was in the anticipated range of 4440 – 8880 Lux within ± 15% from the average.


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: EC10 >0.033 mg/L

Key result

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: EC20 >0.033 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: EC50 >0.033 mg/L

Key result

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: EC10 >0.033 mg/L

Key result

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: EC20 >0.033 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:

other: Yield

Remarks on result:

other: EC50 >0.033 mg/L

Details on results:

The EC values exceeded the water solubility of 0.033 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 Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

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

Yield: 0.31 mg/L
Growth Rate: 0.71 mg/L

Objective

The objective of the present study was to determine the effect of the test item on growth rate and yield of the unicellular green alga Desmodesmus subspicatus.

Study Design

The study comprised of one control group and one test item group with six replicates each. The nominal test item concentration of 100 mg/L was 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 concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (<0.033 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.

No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L.

For the test item the following EC values were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	>0.033 mg/L (nominal >100 mg/L) n.d.	>0.033 mg/L (nominal >100 mg/L) n.d.
EC20 [mg/L] 95% confidence interval	>0.033 mg/L (nominal >100 mg/L) n.d.	>0.033 mg/L (nominal >100 mg/L) n.d.
EC50 [mg/L] 95% confidence interval	>0.033 mg/L (nominal >100 mg/L) n.d.	>0.033 mg/L (nominal >100 mg/L) n.d.

Conclusion

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 72h EC₅₀for growth rate and yield were >0.033 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

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 72h EC50 for growth rate and yield were >0.033 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Executive summary:

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 aquatic toxicity in the test system.The 72h EC₅₀ for growth rate and yield were >0.033 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For this endpoint information from a structural similar compound, 2,3-difluoro-1-methoxy-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexyl]-4-yl]-benzene, is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 201. See chapter 13 report for a more detailed justification.

Reason / purpose for cross-reference:

read-across source

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: EC10 > 0.033 mg/L

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: EC10 > 0.033 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Read Across: (72 h) EC50 > 100 mg/L (nominal)

Executive summary:

For this endpoint information from a structural similar compound, 2,3-difluoro-1-methoxy-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexyl]-4-yl]-benzene, is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 201. See chapter 13 report for a more detailed justification.

Description of key information

Read Across: (72 h) EC50 > 100 mg/L (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

For this endpoint information from a structural similar compound is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 201. See chapter 13 report for a more detailed justification.