4-Ethoxy-2,3-difluorphenylboronic acid

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 5, 2016 - July 15, 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations:
1.0, 2.2, 4.8, 10.6 and 23.4 mg/L

Sampling method:
Samples were taken and analyzed by HPLC from control and test media from both exposure periods, i.e. 0 - 24 hours and 24 - 48 hours. The maintenance of the test item concentration was checked by analysis of freshly prepared samples and after a period of 24 hours.

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

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: 7.83
- Total hardness: 250 mg CaCO3/L (calculated)
- O2-Concentration: 8.36 mg/L

Preparation of the Test Item:
A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, a calibrated flask with test item and vehicle was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was 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 used for the study.

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: 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:

250 mg CaCO3/L (calculated)

Test temperature:

19.6 – 19.9°C

pH:

7.60 - 7.86

Dissolved oxygen:

7.92 - 8.44 mg/L

Nominal and measured concentrations:

Nominal concentrations: 1.0, 2.2, 4.8, 10.6 and 23.4 mg/L
Measured concentrations: > 80 % of nominal concentrations (please refer to "any other information on results")

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
2.2 mg/L: 20 daphnids
4.8 mg/L: 20 daphnids
10.6 mg/L: 20 daphnids
23.4 mg/L: 20 daphnids

CONCENTRATION(S)
Stability determination of the test item in the vehicle revealed no difference between an open and closed system and the recovery rates were >80% of the initial test item concentrations (Broschard, 2016). In a static pre-test a concentration dependent immobilization was seen between 1.0 and 23.4 mg/L.

Therefore, the study was performed as a static test in an open system with the following nominal concentrations:
1.0, 2.2, 4.8, 10.6 and 23.4 mg/L

For the control, reconstituted water (ELENDT M4 medium) was used.

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

- 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 594 Lux and 648 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.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

21.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the nominal concentrations.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the nominal concentrations.

Details on results:

Under the conditions of the present study, the test item showed a 48h EC50 of 7.3 mg/L.

Results with reference substance (positive control):

Potassium dichromate:
24h EC50 0.753 mg/L (0.646 – 0.883 mg/L)
48h EC50 0.602 mg/L (0.525 – 0.689 mg/L)

The test item Art. 104864 (Potassium dichromate) showed a 24h EC50 value of 0.753 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).

Results

	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%
2.2 mg/L	0/20	0/20	0%	0%
4.8 mg/L	0/20	0/20	0%	0%
10.6 mg/L	0/20	19/20	0%	95%
23.4 mg/L	15/20	20/20	75%	100%

	Analytical Results
	0h		24h		48h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%
0 mg/L	< LOD	-	< LOD	-	< LOD	-
1.0 mg/L	0.955	95.5	0.933	93.3	0.939	93.9
2.2 mg/L	2.130	96.8	2.066	93.9	2.091	95.0
4.8 mg/L	4.613	96.1	4.511	94.0	4.541	94.6
10.6 mg/L	10.039	94.7	9.969	94.0	10.068	95.0
23.4 mg/L	21.976	93.9	22.105	94.5	22.139	94.6

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 48h EC50 of 7.3 mg/L to Daphnia magna.

Executive summary:

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 concentrations of the test item (0, 1.0, 2.2, 4.8, 10.6 and 23.4 mg/L) in an open static system. 20 daphnids, divided into four replicates, each with five animals were used per concentration and control. Based on results of a stability determination before start of study, a static test system was chosen. Concentrations of the test item in the test media were analyzed via HPLC with UV detection directly after preparation and then daily. The daphnids were observed for immobilization after 24 and 48 hours of exposure.

Results

The measured concentrations of the test item at the start of exposure were within the range of 93.9% to 96.8% of the nominal concentrations. At the end of the exposure, the measured concentrations were within the range of 93.9% to 95.0% of the nominal concentrations.

As the recovery of the test item in the aqueous solution was >80% during the study, the EC50 values were calculated with the nominal concentrations.

No effect on the mobility of the daphnia was observed at the three lowest concentrations of
1.0 mg/L, 2.2 mg/L and 4.8 mg/L. A concentration dependent immobilization was observed at concentrations of 10.6 and 23.4 mg/L.

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

24h EC50       21.3 mg/L (n.d.)

48h EC50       7.3mg/L (6.4– 8.4 mg/L)

Conclusion

Under the conditions of the present study, the test item showed a 48h EC50 of 7.3 mg/L.

Description of key information

OECD 202: 48h EC50 = 7.3 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

7.3 mg/L

Additional information

The effect of the test item on the immobilization of Daphnia magna was examined in a study according to OECD TG 202.

Juvenile daphnids were exposed to different concentrations of the test item (0, 1.0, 2.2, 4.8, 10.6 and 23.4 mg/L) in an open static system. The daphnids were observed for immobilization after 24 and 48 hours of exposure.

No effect on the mobility of the daphnia was observed at the three lowest concentrations of 1.0 mg/L, 2.2 mg/L and 4.8 mg/L. A concentration dependent immobilization was observed at concentrations of 10.6 and 23.4 mg/L.

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

24h EC50       21.3 mg/L (n.d.)

48h EC50       7.3mg/L (6.4– 8.4 mg/L)