4-Butoxy-2,3-difluorophenylboronic Acid (contains varying amounts of Anhydride)

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:

Apr 03 - Aug 16, 2018

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Two replicates of each sample were deep-frozen (≤ -20°C) directly after preparation and after 24 h, 48 h and 72 h and stored in a freezer until analysis. Before freezing the samples after 24 h, 48 h and 72 h exposure, the algae were separated from the medium by centrifugation at a low g-force (4000 g for 20 minutes), sufficient to settle the algae. Additionally, analysis of all test item concentrations not inoculated with algae were performed. The stability of the test item during freezing has been demonstrated in a separate non-GLP study.

Vehicle:

yes

Remarks:

reconstituted water

Details on test solutions:

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. The pH value was not adjusted.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata (61.81 SAG)
- Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Age of inoculum (at test initiation): three days old
- Method of cultivation: 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers. The cultures were shaken continuously at about 120 rpm and lit between 4440 and 8880 Lux. Fluorescent tubes installed above the flasks served for lighting.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

ca. 0.24 mmol/L (about 25 mg/L CaCO3)

Test temperature:

23.4 to 25.1 °C

pH:

7.78 to 9.14

Details on test conditions:

The study was performed as a static test in open vessels under culture conditions. The flasks contained 100 mL either reconstituted water or test medium (test item group).
The duration of exposure was 72 hours. The algal biomass in each flask was determined daily during the exposure period.

A 3-day old pre-culture, prepared in dilution water, was used as inoculum. Before start of exposure, the cell density was determined. The test vessels were inoculated with an appropriate volumen of algae pre-culture suspension in order to obtain an initial cell density of 10000 cells/mL.

Volumes of the algal suspension not impairing the study were taken from all test item preparations ans the controls after exposure times of 24, 48 and 72 h (+/- 1 h). The required volume depended on the cell density. The withdrawn volumes were not substituted. before the cell density measurement, the algal suspension was diluted and the cell densities were determined with an electronic particle counter. Additionally the blank values without algae were determined at the sampling time points. The respective blank values were substracted from the measured value of each replicate.
The measured cell counts were converted into cell number per mL. To identify the morphological effect of the test item on the algal cells, additional samples were taken from the control and test item group after 72 h. The morphological appearance of the treated algal cells in comparison to the controls was assessed by microscope.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

95% CI:

> 1.63 - < 1.99

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.57 mg/L

95% CI:

> 0.45 - < 0.69

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.83 mg/L

95% CI:

> 0.73 - < 1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.45 mg/L

95% CI:

> 0.36 - < 0.52

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

The recoveries of the test material concentrations in all test media with algae ranged from 42.0 % to 54.2 % from the nominal concentrations over the time period of 72 h. No test item was measured in the control medium (The recoveries of the test material concentrations in all test media without algae ranged from 37.0 % to 55.6 % from the nominal concentrations over the period of 72 h. There was no effect of the presence of algae on the measured test item concentrations in the test media at the different time points. The recoveries from the initial measured concentrations in samples with and without algae ranged from 82.2 % to 101.1 %.

Reported statistics and error estimates:

Under the conditions of the present study, the test item showed a 72 h EC50 of 1.80 mg/L for the growth rate and 0.83 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

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

Nominal test item concentration [mg/L]	Analytical results
	0h^1)		24 h		48 h		72 h
	Meas. Conc. [mg/L]	[%]^2)	Meas. Conc. [mg/L]	[%]^2)	Meas. Conc. [mg/L]	[%]^2)	Meas. Conc. [mg/L]	[%]^2)
0.0	<LOD	--	<LOD	--	<LOD	--	<LOD	--
0.1	0.045	45.0	0.042	42.0	0.045	45.0	0.043	43.0
0.32	0.163	50.9	0.151	47.2	0.147	45.9	0.151^3)	47.2
1.02	0.545	53.4	0.522	51.2	0.513	50.3	0.494	48.4
3.28	1.788	54.5	1.739	53.0	1.710	52.1	1.699	51.8
10.49	5.766	55.0	5.688	54.2	5.563	53.0	5.586	53.3

 

 

LOD = Limit of detection (0.006 mg/L)

¹⁾Measured without algae

²⁾Rounded to the first decimal place

³⁾Mean concentration of two measurements

 

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

Nominal test item concentration [mg/L]	Analytical results
	0 h		24 h		48 h		72 h
	Meas. Conc. [mg/L]	[%]^1)	Meas. Conc. [mg/L]	[%]^1)	Meas. Conc. [mg/L]	[%]^1)	Meas. Conc. [mg/L]	[%]^1)
0.1	0.045	45.0	0.041	41.0	0.038	38.0	0.037	37.0
0.32	0.163	50.9	0.149	46.6	0.149	46.6	0.145	45.3
1.02	0.545	53.4	0.527	51.7	0.520	51.0	0.513	50.3
3.28	1.788	54.5	1.769	53.9	1.795	54.7	1.770	54.0
10.49	5.766	55.0	5.661	54.0	5.705	54.4	5.832	55.6

¹⁾Rounded to the first decimal place

 

EC values:

For the test item, the following EC values were determined:

Parameter (0-72h)	Growth Rate	Yield
EC10[mg/L] 95 % confidence interval	0.57 (0.45 – 0.69)	0.45 (0.36 – 0.52)
EC20[mg/L] 95 % confidence interval	0.85 (0.71 – 0.98)	0.56 (0.48 – 0.63)
EC50[mg/L] 95 % confidence interval	1.80 (1.63 – 1.99)	0.83 (0.73 – 1.00)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 72 h EC50 of 1.80 mg/L for the growth rate and 0.83 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

Description of key information

OECD 201: 72 h EC_r50 = 1.80 mg/L (nominal); 72 h EC_y50 = 0.83 mg/L (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.8 mg/L

EC10 or NOEC for freshwater algae:

0.57 mg/L

Additional information

Under the conditions of the present study, the test item showed a 72 h EC50 of 1.80 mg/L for the growth rate and 0.83 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.