4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

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:

2017-09-11 to 2017-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentration levels and the control
- Sampling method: Samples were taken at the start (0 hours) and at the end of the exposure (72 hours) with algae.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.

Vehicle:

yes

Remarks:

Dilution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal test item concentration of 1 mg/L was freshly prepared with dilution water one day prior to the start of exposure. The saturated solution was stirred for 24 hours at room temperature, 1100 rpm. After a separation phase of 1 hour at room temperature the saturated solution was removed by siphoning (from the approximate center of the glass flask) and used in the test.
- Controls: Potassium dichromate was tested as a reference item.
- Chemical name of vehicle: Dilution water according to the guideline was used.
- Concentration of vehicle in test medium: Medium was used as vehicle.
- Test concentration separation factor: A limit test was done.
- Evidence of undissolved material: The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was observed in the saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 4 days old preculture was used.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2567 - 5130 lux for 24 hours per day. Nutrient medium Z according to LOTTGE et al. (1994) is used.

ACCLIMATION
Acclimatization was not necessary, because the preculture was prepared in dilution water and incubated under test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21.5 - 22.5 °C

pH:

8.02 (start) to 8.93 (end)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

1 mg/L (nominal)
1.67 µg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks
- Type: closed, sealed with cotton wool plugs
- Material, size, headspace, fill volume: glass, size: 250 mL, test volume: 100 mL
- Aeration: no
- Type of flow-through: Not applicable, static
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 6839 cells/mL (actual), approx. 5*10^3-10^4 cells/mL (nominal)
- Control end cells density: mean 1068359 cells/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
- Please refer to “Any other information on methods” for details on medium used.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h/day light
- Light intensity and quality: approx. 4440 to 8880 lux (60 to 120 µE*m^-2*s^-1), homogeneity: within +/- 15 % over incubation area

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the saturated solution.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, limit test
- Justification for using less concentrations than requested by guideline: Limit test based on result of range finder.
- Range finding study
- Test concentrations: 1 mg test item/L was used to prepare saturated solution. 100, 10 an 1 % of saturated solution was used as concentrations in range finding study.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: limit of solubility

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: limit of solubility

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

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: limit of solubility

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: limit of solubility

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: limit of solubility

Results with reference substance (positive control):

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A saturated solution of the test item was used in testing. See details above. The test item concentrations were clear during exposure.
- Effect concentrations exceeding solubility of substance in test medium: No effects were observed up to the solubility limit.

Reported statistics and error estimates:

Growth Rate: EC50=0.559 mg/L
Yield: EC50 =0.320 mg/L

Table 1: Effect concentrations based on geometric mean measured test item concentration [μg/L]

 

	Inhibition of Growth Rate
NOEC	1.67
LOEC	> 1.67
EC10	> 1.67
EC20	> 1.67
EC50	> 1.67
	Inhibition of Yield
NOEC	1.67
LOEC	> 1.67
EC10	> 1.67
EC20	> 1.67
EC50	> 1.67

Validity criteria fulfilled:

yes

Conclusions:

No effects have been detected at the limit of solubility of the test material in the test media. Therefore, the 72h EC50 and 72h EC10 based on growth rate were greater than 1.67 µg/L (geom. mean measured) and considered to correspond to a nominal concentration of 100 mg/L.

Executive summary:

The toxicity of the test material to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 1.00 mg test item/L was prepared once with dilution water 24 hours prior to the start of the exposure. The saturated solution was stirred for 24 hours with approximately 1100 rpm at room temperature. After completion of stirring, the solution was allowed to stand for 1 hour at room temperature for separation of the phases. Thereafter, the saturated solution was removed by siphoning from the approximate centre of the glass flask. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was observed at the saturated solution. Six replicates were used for the limit concentration of the test item and six for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. The concentrations of the test item were analytically verified via GC-MS at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration and the control. The measured concentrations of the limit concentration in the fresh media (0 h) were 11.2 μg/L and in the old media (72 h) 0.249 μg/L. Therefore, all effect values are based on the geometric mean measured test item concentration (1.67 μg/L). No effects of the test item at the limit of solubility were detected and the effect levels based on growth rate were established to be NOEC = 1.67 µg/L (limit of solubility), LOEC > 1.67 µg/L, EC10 > 1.67 µg/L, EC20 > 1.67 µg/L and EC50 > 1.67 µg/L,  thus corresponding to 100 mg/L nominal concentrations.

Description of key information

No effects have been detected up to the limit of solubility of the test material in the test media. Therefore, the 72h EC50 and 72h EC10 based on growth rate were greater than 1.67 µg/L (geom. mean measured) and considered to correspond to a nominal concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The toxicity of the test material to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 1.00 mg test item/L was prepared once with dilution water 24 hours prior to the start of the exposure. The saturated solution was stirred for 24 hours with approximately 1100 rpm at room temperature. After completion of stirring, the solution was allowed to stand for 1 hour at room temperature for separation of the phases. Thereafter, the saturated solution was removed by siphoning from the approximate centre of the glass flask. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was observed at the saturated solution. Six replicates were used for the limit concentration of the test item and six for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. The concentrations of the test item were analytically verified via GC-MS at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration and the control. The measured concentrations of the limit concentration in the fresh media (0 h) were 11.2 μg/L and in the old media (72 h) 0.249 μg/L. Therefore, all effect values are based on the geometric mean measured test item concentration (1.67 μg/L). No effects of the test item at the limit of solubility were detected and the effect levels based on growth rate were established to be NOEC = 1.67 µg/L (limit of solubility), LOEC > 1.67 µg/L, EC10 > 1.67 µg/L, EC20 > 1.67 µg/L and EC50 > 1.67 µg/L, thus corresponding to 100 mg/L nominal concentrations.