4-Ethoxy-2,3-difluorphenylboronic acid

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:

June 10, 2016 - January 9, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations:
0.5, 1.0, 2.0, 4.0 and 8.0 mg/L

Sampling method:
Samples of the test medium and the control medium were taken directly after preparation and after 24, 48 and 72 hours and were subjected to chemical analysis. Before analysis, the algae were separated from the medium by centrifugation at a low g-force (4000g for 20 minutes), sufficient to settle the algae. Additionally, analysis of the test item in all test media preparations without algae was performed daily.

Sample storage conditions before analysis:
Samples were directly analyzed without storing

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: 7.85
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
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 treated in an ultrasonic device for 30 minutes and subsequently stirred with a magnetic stirrer for further 23 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was diluted with the vehicle to the different test item concentrations.

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 was Desmodesmus subspicatus, strain 86.81 SAG (Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany).
- 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:

no

Total exposure duration:

72 h

Post exposure observation period:

-

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

23.6 - 24.2°C

pH:

7.74 - 8.44

Nominal and measured concentrations:

Nominal concentrations: 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L
Measured concentrations: >80% of nominal concentrations (please refer to "any other information on results"

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: 1 126 608 cells per ml
- No. of vessels per concentration (replicates): 3
- 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 mean light intensities were 8314 Lux ± 4.0% and 8328 Lux ± 4.9% prior to and at the end of the exposure period, respectively.
The mean light intensities for the samples used for analytical determination after 24 and 48 hours were 8337 Lux ± 2.9% and 8406 Lux ± 2.3% prior to and at the end of the exposure period, respectively.

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0.5, 1.0, 2.0, 4.0 and 8.0 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 (Gado, 2016).

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.028 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC10 were calculated based on nominal concentrations

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.359 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC20 were calculated based on nominal concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC50 were calculated based on nominal concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.509 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC10 were calculated based on nominal concentrations

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.736 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC20 were calculated based on nominal concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.269 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC50 were calculated based on nominal concentrations

Details on results:

- 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 (Gado, 2016).

The test item Art. 104864 (Potassium dichromate) showed a 72h EC50 of 0.72 mg/L for growth rate and 0.40 mg/L for biomass increase. These values are within the recommended ranges for growth rate (EC50 0.60 – 1.03 mg/L) and biomass integral (EC50 0.20 – 0.75 mg/L).

Results

Nominal and corresponding analytical concentrations of samples inoculated with algae are given in the following table:

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.0 mg/L	<LOD	-	<LOD	-	<LOD	-	<LOD	-
0.5 mg/L	0.433	86.6	0.418	83.6	0.414	82.8	0.402	80.4
1.0 mg/L	0.877	88.7	0.847	84.7	0.846	84.6	0.843	84.3
2.0 mg/L	1.763	88.2	1.704	85.2	1.696	84.8	1.692	84.6
4.0 mg/L	3.498	87.5	3.469	86.7	3.484	87.1	3.468	86.7
8.0 mg/L	7.051	88.1	6.810	85.1	6.836	85.5	6.893	86.2

Nominal and corresponding concentrations of analytical control samples without algae are given in the following table:

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.5 mg/L	0.433	86.6	0.427	85.4	0.413	82.6	0.410	82.0
1.0 mg/L	0.877	88.7	0.872	87.2	0.862	86.2	0.855	85.5
2.0 mg/L	1.763	88.2	1.727	86.4	1.701	85.1	1.723	86.2
4.0 mg/L	3.498	87.5	3.499	87.5	3.514	87.9	3.473	86.8
8.0 mg/L	7.051	88.1	7.093	88.7	6.821	85.3	6.950	86.9

For the test item the following EC values were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	2.028 (1.830 - 2.195)	1.509 (0.555 - 1.773)
EC20 [mg/L] 95% confidence interval	2.359 (2.177 - 2.513)	1.736 (0.968 - 1.957)
EC50 [mg/L] 95% confidence interval	3.148 (3.003 - 3.288)	2.269 (2.025 - 3.276)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 72h EC50 of 3.148 mg/L and a 72h EC10 of 2.028 mg/L for growth rate.

Executive summary:

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

The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 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.

The concentrations of the test item were analytically determined by HPLC-UV. Analytical controls of the test item preparations were performed at the start of the study and then daily.

The recoveries of the test item concentrations in all test media inoculated with algae and without algae ranged from 80.4% to 88.7% from the nominal concentrations over the time period of 72h.

As the recoveries of the test item in the test media were >80.0% during the study, the EC50 values were calculated based on the nominal concentrations.

For the test item the following EC values for growth rate were determined after 72 hours:

EC10 = 2.028 g/L (95% CI: 1.830 - 2.195 mg/L)

EC20 = 2.359 mg/L (95% CI: 2.177 - 2.513 mg/L)

EC50 = 3.148 mg/L (95% CI: 3.003 - 3.288 mg/L)

Under the conditions of the present study, the test item showed a 72h EC50 of 3.148 mg/L and a 72h EC10 of 2.028 mg/L for growth rate.

Description of key information

OECD 201:

72h EC50 = 3.148 mg/L based on growth rate

72 h EC10 = 2.028 mg/L based on growth rate

Key value for chemical safety assessment

EC50 for freshwater algae:

3.148 mg/L

EC10 or NOEC for freshwater algae:

2.028 mg/L

Additional information

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

The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 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.

The concentrations of the test item were analytically determined by HPLC-UV. Analytical controls of the test item preparations were performed at the start of the study and then daily.

The recoveries of the test item concentrations in all test media inoculated with algae and without algae ranged from 80.4% to 88.7% from the nominal concentrations over the time period of 72h.

As the recoveries of the test item in the test media were >80.0% during the study, the EC50 values were calculated based on the nominal concentrations.

For the test item the following EC values for growth rate were determined after 72 hours:

EC10 = 2.028 g/L (95% CI: 1.830 - 2.195 mg/L)

EC20 = 2.359 mg/L (95% CI: 2.177 - 2.513 mg/L)

EC50 = 3.148 mg/L (95% CI: 3.003 - 3.288 mg/L)

Under the conditions of the present study, the test item showed a 72h EC50 of 3.148 mg/L and a 72h EC10 of 2.028 mg/L for growth rate.