4'-Ethyl-3-fluorbiphenyl-4-boronic acid

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

jun 04, 2019 - Aug 22, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test medium (reconstituted water and test item) was prepared freshly. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

Analytical monitoring:

yes

Details on sampling:

The present study was performed as a semi-static test with a renewal of the test item preparations (test item groups) and the vehicle (control group) every 24 hours. Analysis of the test item concentration directly after each preparation (fresh medium) and after 24h (old medium) were performed. Two replicates of each sample were deep-frozen (≤ -20°C) and stored in a freezer until analysis. One replicate was given to the analytical laboratory for analysis.

Vehicle:

yes

Details on test solutions:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test medium (reconstituted water and test item) was prepared freshly. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

VEHICLE:
For the reconstituted water the following chemicals (analytical grade) were dissolved in fully demineralized water:

Macro nutrients mg/L
CaCl2 x 2H2O 294.00
MgSO4 x 7H2O 123.25
NaHCO3 64.75
KCl 5.75


Before the start of the experimental part the reconstituted water for fish was prepared. After preparation the water was aerated for 24 hours before using it.
The proportion of Ca to Mg ions was 4:1 and that of Na to K ions 10:1. Hardness: about 228 mg/L CaCO3.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Dania rerio
- Origin: Max-Planck-Institut, Tübingen, Germany
- Breeder: Chemical & Preclinical Safety, Merck KGaA Darmstadt, Germany
- Length at study initiation: 2.6 - 2.6 cm
- Weight at study initiation: 0.3 - 0.4 g
- Feeding during test: commercial fish diet during acclimatization

ACCLIMATION
- Acclimation period: The fish were acclimatized to the conditions of the laboratory for more than 14 days and were held in reconstituted water for at least 12 days before testing.
- Acclimation conditions (same as test or not): same
- Health during acclimation (any mortality observed): no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

228 mg/L CaCO3

Test temperature:

20.5 to 21.5°C

pH:

7.49 to 8.00

Dissolved oxygen:

72.8 - 93.1%

Conductivity:

710 µS/cm

Nominal and measured concentrations:

Concentrations in mg/L
nominal measured (0h) measured (24 h, old) measured (24 h, new) measured (48h, old)
5.0, 0.106 0.078 0.119 0.102
10.6 0.221 0.164 0.256 0.200
22.4 0.483 0.377 0.555 0.461
47.3 1.028 0.761 1.266 0.969
100.0 1.816 1.571 2.233** 2.232**

** not used in calculation as all fish were already death

Details on test conditions:

TEST SYSTEM
- Test vessel: 10L Glass Aquariums
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Full glas folled with 10L test medium
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according OECD 203
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides:no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: 4/1
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12hrs light / 12hrs dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality, clinical symptoms

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.765 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The test material, dissolved in reconstituted water, was tested in an open semi-static system. Under the conditions of the present study, the 96h LC50 was 0.765 mg/L.


The 96h LC50 exceeded the water solubility of 0.0000001448 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.


- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes

Sublethal observations / clinical signs:

Mortality was observed in the two highest concentrations tested (i.e. nominal 47.3 and 100.0 mg/L). No mortality was seen in the nominal concentrations 5.0 up to 22.4 mg/L and in the control group.

Nominal Test Item Concentration [mg/L]	Number of dead fish / exposed
	3 hours	24 hours	48 hours	72 hours	96 hours
0	0/7	0/7	0/7	0/7	0/7
5.0	0/7	0/7	0/7	0/7	0/7
10.6	0/7	0/7	0/7	0/7	0/7
22.4	0/7	0/7	0/7	0/7	0/7
47.3	0/7	0/7	1/7	4/7	7/7
100.0	0/7	7/7	7/7	7/7	7/7

No clinical symptoms were observed in the lowest test item concentrations groups (i.e. nominal 5.0 and 10.6 mg/L) and the control group. Fish near the bottom of the aquarium were observed at nominal concentrations from 22.4 mg/L up to nominal 100.0 mg/L. Additionally, discoloration (light) was seen at a nominal concentration of 47.3 mg/L.

Nominal Test Item Concentration [mg/L]	Clinical Symptom	Number of fish with symptoms / exposed
		3 hours	24 hours	48 hours	72 hours	96 hours
0	No symptoms	7/7	7/7	7/7	7/7	7/7
5.0	No symptoms	7/7	7/7	7/7	7/7	7/7
10.6	No symptoms	7/7	7/7	7/7	7/7	7/7
22.4	No symptoms	7/7	7/7	6/7	5/7	2/7
	Fish near the bottom of the aquarium			1/7	2/7	5/7
47.3	No symptoms	7/7	1/7	0/6	0/3	-
	Fish near the bottom of the aquarium		6/7	6/6	3/3	-
	Discoloration (light)		3/7	3/6	2/3	-
100.0	Fish near the bottom of the aquarium	7/7	-	-	-	-

A maximum stocking density of 1g fish per liter test medium was not exceeded and length was within the range of 2.0 ± 1.0 cm.

Nominal Test Item Concentration [mg/L]	Length	Body Weight
Control	2.6 – 2.8 cm	0.3 – 0.4 g
5.0	2.7 – 2.8 cm	0.3 – 0.4 g
10.6	2.6 – 2.8 cm	0.3 – 0.4 g
22.4	2.7 – 2.8 cm	0.3 – 0.4 g
47.3	2.6 – 2.8 cm	0.3 – 0.5 g
100.0	2.6 – 2.9 cm	0.3 – 0.5 g

The pH-value ranged from 7.69 to 7.98.

Nominal concentration [mg/L]	0 hours fresh	24 hours old	24 hours fresh	48 hours old	48 hours fresh	72 hours old	72 hours fresh	96 hours old
Control	7.98	7.69	7.94	7.70	7.89	7.74	7.91	7.71
5.0	7.85	7.72	7.95	7.73	7.91	7.75	7.93	7.72
10.6	7.93	7.72	7.95	7.75	7.92	7.75	7.94	7.73
22.4	7.95	7.74	7.95	7.76	7.93	7.73	7.94	7.73
47.3	7.96	7.71	7.96	7.73	7.94	7.69	7.95	7.70
100.0	7.98	7.71	7.97	7.90	7.95	7.90	7.96	7.94

The oxygen concentrations were within normal range.

Nominal concentration [mg/L]	O2[%]
	0 hours fresh	24 hours old	24 hours fresh	48 hours old	48 hours fresh	72 hours old	72 hours fresh	96 hours old
Control	93.0	89.3	93.3	88.6	92.6	91.6	94.7	90.1
5.0	95.1	90.4	94.2	89.9	94.3	91.2	94.7	90.9
10.6	94.7	88.6	93.9	89.1	94.0	90.6	94.7	89.3
22.4	94.1	90.6	92.1	88.7	93.9	90.1	94.0	88.9
47.3	93.8	87.6	90.5	90.1	93.6	88.6	92.1	90.1
100.0	75.7	82.4	71.2	84.0	72.9	85.1	71.9	83.1

The water temperature (0-96 hours) ranged from 21.6 to 24.5°C. The water temperature was transiently outside the anticipated range. This minor transient deviation did not influence the integrity or outcome of the study.

Validity criteria fulfilled:

yes

Conclusions:

Under the semi-static conditions of the present study, the test material, dissolved in reconstituted water showed a 96h LC50 of 0.765 mg/L.

Executive summary:

The objective of this study was to detemiine the acute toxicity of the test item in zebrafish (Danio rerio). Zebrafish were exposed to 5 different concentrations of the test item (5.0, 10.6, 22.4, 47.3 and

100.0 mg/L) prepared in reconstituted water and a control group in an open semi-static system. Seven zebrafish were used in the test item groups and control group, respectively. The test item preparations (test item groups) and the vehicle (control group) were renewed every 24 hours. The zebrafish were observed for signs of toxicity and mortality for 96 hours. Samples of the test media and control medium were taken directly (fresh medium) and 24h ( old medium) after each preparation and stored at <= -20°C until further analysis of the test item concentration via HPLC with UV-detection.

Under the semi-static conditions of the present study, the test material, dissolved in reconstituted water showed a 96h LC50 of 0.765 mg/L.

Description of key information

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 203. Under the semi-static conditions of the present study, the test material, dissolved in reconstituted water showed a 96h LC50 of 0.765 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.765 g/L