4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

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:

2017-08-27 to 2017-08-31

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:

7 December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

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 (0.00306 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.

Vehicle:

yes

Remarks:

Reconstituted water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. Th pH value was not adjusted.
- Controls: Vehicle control was done.
- Chemical name of vehicle: Reconstituted water
- Concentration of vehicle in test medium: Not applicable as test medium was used as vehicle.
- Test concentration separation factor: Not applicable as limit test was done.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No. The test media were clear preparations and stayed unchanged throughout the study.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata strain 61.81 SAG
- Source (laboratory, culture collection): obtained from „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany
- Age of inoculum (at test initiation): 3 days old pre-culture was used
- 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: not applicable as same conditions applied
- Culturing media and conditions: 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

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

23.9 - 24.5 °C

pH:

7.76 - 9.34

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentration: 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: 10000 cells per mL
- Control end cells density: 3409647 cells per mL (mean)
- 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. For details on composition please refer to “Any other information on materials and method”.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The mean light intensities were 7674 Lux ± 5.2 % and 7481 Lux ± 4.1 % prior to and at the end of the exposure period, respectively.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter (after 24 ± 1 h, 48 ± 1 h, 72 ± 1 h)
- Other: The morphological appearance of the treated algal cells in comparison to the controls was assessed by microscope after 72 h exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable as limit test was done
- Range finding study: pre-test was conducted
- Test concentrations: 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: No effects were observed at 100 mg/L (nominal) thus, the limit test was conducted.

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 was tested as positive control in a separate study.

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: > 0.00306 mg/L (limit of solubility)

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: > 0.00306 mg/L (limit of solubility)

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: > 0.00306 mg/L (limit of solubility)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.00306 mg/L (limit of solubility)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.00306 mg/L (limit of solubility)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.00306 mg/L (limit of solubility)

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 0.00306 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 morphological changes of the treated algae in comparison to the control algae were observed.
- Unusual cell shape: No morphological changes of the treated algae in comparison to the control algae were observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test media were clear preparations and stayed unchanged throughout the study.

Results with reference substance (positive control):

A separate study was conducted to demonstrate the accuracy and reliability of the test method using potassium dichromate as positive control. The study comprised of one control group with six replicates and five potassium dichromate groups with three replicates each. The 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 10000 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. Potassium dichromate showed 72h EC50 of 0.86 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means. Therefore, this study confirmed the sensitivity and reliability of the test system used in the test facility.

Validity criteria fulfilled:

yes

Conclusions:

No adverse effects on algae were observed up to the water solubility of the test item. Thus, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 and EC10 values were > 0.00306 mg/L (nominal > 100 mg/L).

Executive summary:

The present study was conducted according to OECD TG 201 to determine the effect of the test item on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in 100 mL reconstituted water. The initial algae cell density in each replicate was 10000 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. 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 (0.00306 mg/L), the test item 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 rate and yield of the algae were observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

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

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.
EC20 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.
EC50 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.

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 EC10 values for growth rate and yield were >0.00306 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Description of key information

No adverse effects on algae were observed up to the water solubility of the test item. Thus, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 and EC10 values were > 0.00306 mg/L (nominal > 100 mg/L) (reference 6.1.5-1).

Additional information

The present study was conducted according to OECD TG 201 to determine the effect of the test item on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in 100 mL reconstituted water. The initial algae cell density in each replicate was 10000 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. 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 (0.00306 mg/L), the test item 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 rate and yield of the algae were observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

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

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.
EC20 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.
EC50 [mg/L] 95 % confidence interval	>0.00306 mg/L (nominal >100 mg/L) n.d.	>0.00306 mg/L (nominal >100 mg/L) n.d.

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 EC10 values for growth rate and yield were >0.00306 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.