3A-Flux

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 10, 2021 - July 06, 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

18. June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium hydrogen fluoride, reaction product with boric acid and dipotassium salt of boric acid

Cas Number:

2787482-26-0

Molecular formula:

not applicable (UVCB)

IUPAC Name:

Potassium hydrogen fluoride, reaction product with boric acid and dipotassium salt of boric acid

Test material form:

solid - liquid: suspension

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: provided by sponsor, batch no. K-629-D
- Purity, including information on contaminants, isomers, etc.: Boric acid (H2B4O7), dipotassium salt, reaction products with boron and potassium fluoride (K(HF2)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20 ± 5 °C); keep dry
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable under storage conditions, homogeneous – during storage, phase-formation might occur; before use always homogenize by gently stirring, stability in solvents unknown
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Solubility H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown; Stability unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): gentle stirring for homogeneity
- Final concentration of a dissolved solid, stock liquid or gel: stock solution containing 1001 mg/L in test medium was prepared. The concentration to be tested was prepared by dilution of this stock solution with test medium.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: pH was measured (3.6) and adjusted to pH 7.0 using sodium hydroxide (1m)

Sampling and analysis

Analytical monitoring:

not required

Details on sampling:

- Concentrations: 1-7 mg/L B and K (steps of 1mg/L)
- Sampling method: measurement of B, K and F at beginning and end of test, recording of mortality after 1, 4 and 24h after start of exposure, on days 2-4 all vessels were inspected twice daily. Based on the measured concentrations of the three components and results of characterization of the test item, the test item concentration was calculated (see details on test solutions)

Test solutions

Details on test solutions:

DETAILS ON PRETREATMENT
- Dilution: for the measurement of K, the test item solution was diluted tenfold, the others remained undiluted.

METHOD BORON, POTASSIUM:
- Detection method (e.g. ECD, UV, MS, ICP-AES, ICP-MS): ICP-OES
- Detection limits (LOD, LOQ) (indicate method of determination/calculation): The lowest calibration level calculated with sufficient precision was stated as the LOQ (limit of quantification). The limit of detection (LOD) was calculated as follows:
LOD = LOQ / 3. LODs: 0.3 mg/L B and K
- Specificity: checked by measuring the blank value of calibration solvent reconstituted freshwater in duplicate
- Calibration in reconsituted freshwater (FW): range of 1 – 7 mg/L B and K in reconstituted freshwater was chosen. Solutions of the concentrations 1 / 2 / 3 / 4 / 5 / 6 / 7 mg/L were prepared each by dilution of 100 mg/L stock solutions with reconstituted freshwater. The 100 mg/L stock solutions were prepared from a certified stock solutions B and K (concentration 1000 mg/L, nominal, each). To all calibration solutions, 1000 μL HNO3 (65 %) was added and the mixtures were filled up to 100 mL with reconstituted freshwater
- Applicability of Calibration: Directly after calibration and on the subsequent sampling days, QC (quality control) samples from the calibrated range 4 mg/L B and K were measured in order to verify the applicability of the calibrations
- Accuracy and Precision: Accuracy and precision in test medium were demonstrated at the following fortification levels: 1 mg/L and 7 mg/L B and K in test medium. Five replicates of each fortification level were prepared analogously to the preparation of the calibration levels (using the stock solution 100 mg/L). Two QC samples were measured two times. The condition "RSD≤20 %per used level" was fulfilled

METHOD FLUORIDE:
- Detection method (e.g. ECD, UV, MS, ICP-AES, ICP-MS): IC-determination
- Sample preparation: no dilution
- Specificty: The specificity was checked by measurement 2 replicates of the blank value of calibration solvent reconstituted freshwater in duplicate. No interfering signal were detected
- Calibration in reconsituted freshwater (FW): range of 5 – 50 mg/L F in reconstituted freshwater was chosen. Solutions of the concentrations 5 / 10 / 15 / 20 / 25 / 30 / 45 / 50 mg/L were prepared by dilution of a 100 mg/L stock solution with reconstituted freshwater. The 100 mg/L stock solution was prepared from a certified stock solution F (concentration 1000 mg/L, nominal).
- Applicability of calibration: Directly after calibration and on the subsequent sampling days, QC (quality control) samples from the calibrated range 25 mg/L F were measured in order to verify the applicability of the calibrations
_ Accuracy and precision: in test medium were demonstrated at the following fortification levels: 10 mg/L and 45 mg/L F- in test medium. Five replicates of each fortification level were prepared analogously to the preparation of the calibration levels (using the stock solution 100 mg/L)

CHARACTERIZATION:
- Characterization: by weighting of 50 ± 5 mg test item in 50 mL demineralised water and analysis of the test item solution for B, K and F after dilution in order to get the values in calibration range. The determination was performed in triplicate. As B and K are present in reconstituted freshwater, a new calibration in demineralized water was performed for the characterization of the test item.
For the determination of F during the characterization, the calibration in reconstituted freshwater was used, as F is not present in this medium.

CALIBRATION B AND K IN DEMINERALIZED WATER:
- Calibration: range of 1 – 7 mg/L B and K in demineralized water was chosen. Solutions of the concentrations 1 / 2 / 3 / 4 / 5 / 6 / 7 mg/L were prepared each by dilution of 100 mg/L stock solutions with demineralized water. The 100 mg/L stock solutions were prepared from a certified stock solutions B and K (concentration 1000 mg/L, nominal, each). To all calibration solutions, 1000 μL HNO3 (65 %) was added and the mixtures were filled up to 100 mL with demineralized water.

All Results are attached as background material.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: originally from University Heidelberg (2019), offspring was used in test
- Age at study initiation (mean and range, SD): sexually immature young fish of the same age
- Length at study initiation (length definition, mean, range and SD):1 – 2 cm
- Weight at study initiation (mean and range, SD):
- Maintenance of the brood fish: following SOP 115 005 04 edition 9, valid from 26. Apr. 2021 with the following conditions: Medium reconstituted freshwater,
Food one to two times each working day; warm-water fish food and artemia,
Photo period 12/12 hours using LED’s, Temperature 23 ± 2 °C

ACCLIMATION
- Acclimation period: not specified, at least 24h
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: warm-water fish food and artemia
- Feeding frequency during acclimation: 1-2 times on working days
- Health during acclimation (any mortality observed): mortality was belog 5% during the last 7 days before testing

Study design

Test type:

static

Water media type:

other: reconstituted freshwater

Remarks:

tap water is desalinated by reverse osmosis, conductivity and pH value are then adjusted by adding electrolyte solutions (sea salt and sodium bicarbonate)

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

53.4 mg CaCO3/L

Test temperature:

in °C, hours 0 - 24 - 48 - 72 - 96:
blank control: 24.0 - 23.5 - 24.4 - 23.5 - 23.7
in test concentration: 24.0 - 23.4 - 24.4 - 23.4 - 23.6

pH:

hours 0 - 24 - 48 - 72 - 96:
blank control: 7.7 - 7.9 - 7.8 - 7.7 - 7.8
in test concentration: 7.6 - 7.8 - 7.7 - 7.7 - 7.7

Dissolved oxygen:

O2 values in mg/L, at hours 0 - 24 - 48 - 72 - 96:
blank control: 7.9 - 8.1 - 8.2 - 8.0 - 8.0
in test concentration: 8.0 - 8.2 - 8.2 - 7.9 - 7.8

Nominal and measured concentrations:

nominal: 100mg/L
measured: based on all components at beginning in range between 101.2 - 110.6% and after 96h 104.2 - 116.1%

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria, max volume 12L
- Aeration: accomplished with glass tubes
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 7
- Biomass loading rate: 1 fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted freshwater, see "Water media type"
- Total organic carbon: 0.07 mg/L
- Intervals of water quality measurement: measured at beginning (Hardness, Nitrate, TOC) and every 24 ± 1h (O2, Temperature, pH)

OTHER TEST CONDITIONS
- Adjustment of pH: yes, see "Water media type"
- Photoperiod: 12/12 h using LEDs
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): contents of Boron, Potassium and Fluoride (as components of the test item) at beginning and end of test

TEST CONCENTRATIONS
- Test concentrations: 100mg/L
- Results used to determine the conditions for the definitive study: The measured concentrations based on all components at the beginning of the test were in range between 101.2 % and 110.6 % of the nominal concentration and at the end of the test in a range between 104.2 % and 116.1 % of the nominal concentration. Therefore, the de-termination of the biological result was based on the nominal concentration. The following result for the test item 3A-Flux could be determined:
96h-LC50 > 100 mg/L

Results and discussion

Effect concentrations

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: none
- Observations on body length and weight: see Table 1 in "Any other information" in Test conditions
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured concentrations based on all components at the beginning of the test were in range between 101.2 % and 110.6 % of the nominal concentration and after 96 hours in a range between 104.2 % and 116.1 % of the nominal concentration. Therefore, the determination of the biological result was based on the nominal concentration. (see OECD Guid-ance Doc. No.23 (§176)).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this valid experiment, the measured concentrations based on all components at the beginning of the test were in range between 101.2 % and 110.6 % of the nominal concentration and after 96 hours in a range between 104.2 % and 116.1 % of the nominal concentration. Therefore, the determination of the biological result was based on the nominal concentration.
The 96h LC50 was determined >100 mg/L.

Executive summary:

One valid experiment in which the toxicity against Danio rerio was tested in a static design was performed. The limit test was performed at the concentration of 100mg/L (nominal) and showed no mortality in both treatment and control. In the beginning and the end of the test, Boron, Potassium and Fluroide were determined using ICP-OES-determination (B, K) and IC-determination (F) respectively.

Based on the measured concentrations and the results of the characterisation of the test item, the test item concentration was calculated.