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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 3, 2018 - July 5, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation (EC) No. 440/2008 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)

Specific details on test material used for the study:

Preparation of the Test Item:
A 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 preparation of the different concentrations. The pH was not adjusted.

Analytical monitoring:

yes

Details on sampling:

Concentrations:
1.0, 1.8, 3.2, 5.8 and 10.5 mg/L

Sampling method:
Two replicates of each sample were deep-frozen (≤ -20 °C) directly after preparation and after 48h and stored in a freezer until analysis. The stability of the test item during freezing has been demonstrated in a separate non-GLP study. One replicate was used for analysis. The second replicate was used as backup.

Vehicle:

yes

Details on test solutions:

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.82
- Total hardness: 232 mg CaCO3/L
- O2-Concentration: 8.76 mg/L
- Conductivity: 700 µS/cm

Preparation of the Test Item:
A 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 preparation of the different concentrations. The pH was not adjusted.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna Straus
Origin: Daphnia magna Straus was originally obtained from IBACON GmbH (Roßdorf, Germany).
Culture conditions: The clone is bred in the laboratories of Merck KGaA (room 2068).
Parental daphnids are used for reproduction until they are about 6 weeks old. Thereafter, they are replaced by neonates.
Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2°C and a 16 hour light and 8 hour dark regime to ensure similar conditions as in the experiment. Offspring are removed from the vessels at least twice per week.
Feeding: The parental daphnids are fed ad libitum with unicellular green algae Desmodesmus subspicatus three times per week.
Age: Offspring less than 24 hours old were used for the study.
Acclimation period: same as test

Feeding during test: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

232 mg CaCO3/L

Test temperature:

20.2 - 20.8 °C

pH:

7.76 - 7.90

Dissolved oxygen:

8.26 - 8.64 mg/L

Conductivity:

700 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 1.0, 1.8, 3.2, 5.8 and 10.5 mg/L
Measured concentrations:The measured concentrations of the test item were between 54.2 % and 55.3 % of the nominal concentrations at 0 hours with recovery rates between 96.1 % and 99.4 % after 48 hours.

Details on test conditions:

EXPOSURE:
The study was performed in an air-conditioned room. For the study 20 mL glass test tubes containing at least 20 mL either reconstituted water (control group) or test medium (test item groups) were used. Each test vessel contained five daphnids resulting in 4 mL medium per daphnid. They were not fed and the media were not aerated during the exposure.

The test was performed as a static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
1.0 mg/L: 20 daphnids
1.8 mg/L: 20 daphnids
3.2 mg/L: 20 daphnids
5.8 mg/L: 20 daphnids
10.5 mg/L: 20 daphnids

CONCENTRATION(S)
In a pre-test (non-GLP) the stability of the test item in reconstituted water for daphnids was investigated. The solubility of the test item in the test medium was approx. 60% from the nominal concentration. However, the test item concentration in reconstituted water for daphnids was stable over a period of 48 hours in open vessels and that samples can be deep-frozen until analysis (Beranek, 2018).

Therefore, the present study was performed as a static test in open vessels with the following nominal concentrations:

1.0, 1.8, 3.2, 5.8 and 10.5 mg/L

For the control, reconstituted water was used.


VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.82
- Total hardness: 232 mg CaCO3/L
- O2-Concentration: 8.76 mg/L
- Conductivity: 700 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 48h


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark
- Light intensity: The mean light intensities were 510 Lux and 537 Lux prior to and at the end of the exposure period, respectively.

References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990

Reference substance (positive control):

no

Remarks:

No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the initial measured concentrations.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the initial measured concentrations.

Details on results:

Under the conditions of the present study, the test item showed a 48 h EC50 of 1.3 mg/L.

Results with reference substance (positive control):

Potassium dichromate:
24 h EC50 1.08 mg/L (0.94 – 1.27 mg/L)
48 h EC50 0.76 mg/L (0.67 – 0.86 mg/L)

The test item Art. 104864 (Potassium dichromate) showed a 24h EC50 value of 1.08 mg/L which is within the range of the published data of 0.6 to 1.7 mg/L (Council Regulation (EC) No. 440/2008) and 0.6 to 2.1 mg/L (OECD Guideline No. 202).

Objective

The objective of this study was to determine the effect of the test item on the immobilization of Daphnia magna.

 

Study Design

Juvenile daphnids were exposed to different test item concentrations of nominal 0.0, 1.0, 1.8, 3.2, 5.8 and 10.5 mg/L in an open static system. 20 daphnids, divided into four replicates, each of five animals were used per test item group and the control group. The daphnids were observed for immobilization after 24 and 48 hours of exposure.

 

Samples of the test and control medium were taken directly after preparation and at the end of exposure (48 h) and stored at ≤ -20 °C until further analysis of the test item concentration via HPLC with UV detection.

 

Results

The measured concentrations were between 54.2 % and 55.3 % of the nominal concentrations at 0 hours with recovery rates between 96.1 % and 99.4 % after 48 hours. No test item was measured in the control medium therefore, it was confirmed that daphnia in the control group were not exposed to the test item.

 

	0h		48h
Nominal	[mg/L]	%	[mg/L]	%
0 mg/L	<LOD	-	<LOD	-
1.0 mg/L	0.542	54.2	0.521	52.1
1.8 mg/L	0.994	55.2	0.974	54.1
3.2 mg/L	1.769	55.3	1.743	54.5
5.8 mg/L	3.194	55.1	3.174	54.7
10.5 mg/L	5.773	55.0	5.730	54.6

 

As the recoveries of the test item in the aqueous solutions were >80 % of the initial measured concentrations during the study, the EC₅₀ values were calculated with the initial measured concentrations.

 

No effect on the mobility of the daphnia was observed at the lowest concentrations of nominal 1.0 mg/L. A concentration dependent immobilization was observed between 1.8 and 10.5 mg/L as given in the following table.

 

	Number of daphnids immobilized / exposed		% of daphnids immobilized
Nominal	24 hours	48 hours	24 hours	48 hours
0.0 mg/L	0/20	0/20	0%	0%
1.0 mg/L	0/20	0/20	0%	0%
1.8 mg/L	0/20	3/20	0%	15%
3.2 mg/L	5/20	18/20	25%	90%
5.8 mg/L	7/20	20/20	35%	100%
10.5 mg/L	17/20	20/20	85%	100%

 

For the test item the following nominal EC₅₀ values and 95 % confidence intervals were determined:

24 h EC50       3.3 mg/L (2.7 - 4.3 mg/L)

48 h EC50       1.3 mg/L (1.1 - 1.5 mg/L)

 

Conclusion

Under the conditions of the present study, the test item showed a 48 h EC₅₀ of 1.3 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 48 h EC50 of 1.3 mg/L.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 202. Under the conditions of the present study, the test item showed a 48h EC50 of 1.3 mg/L.

Description of key information

OECD 202: 48 h EC50 1.3 mg/L (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

1.3 mg/L

Additional information

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 202.Under the conditions of the present study, the test item showed a 48h EC50of 1.3 mg/L.