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Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
GLP compliance:
no
Remarks:
Published study not conducted to GLP
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dissolved and stored in ethanol
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stock solution prepared at 10 mg/mL. Stability of the test item in ethanol not reported.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in ethanol to form a stock solution of 10 mg/mL concentration.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 10 mg/mL
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: n/a
Radiolabelling:
no
Remarks:
Deuterated BPAF used to prepare linear response curve for HPLC analysis
Details on sampling:
- Sampling intervals/frequency for test organisms:

Test 1: 12 fish (6 male and 6 female) from each exposure group sampled at 168 h.

Test 2: 4 fish (2 male and 2 female) from each tank (16 fish in total, as there were 4 replicates) sampled at 0, 3, 6, 12, 24, 72, 120 and 168 h during the uptake phase and 0, 2, 4,6 and 24 h during the depuration phase.

- Sampling intervals/frequency for test medium samples:

Test 1: Not reported

Test 2: At each fish sampling interval

- Sample storage conditions before analysis: Not reported
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):

Test 1: The whole-body fish were suspended in 1 mL acetonitrile and homogenized at a vertical velocity of 6 m/s for 40 s or longer using a FastPrep-24 Tissue and Cell Homogenizer (MP Biomedicals, Santa Ana, California, USA). This procedure resulted in complete homogenization of the whole-body fish. After sonication for 10 min and centrifugation at 12,000 g for 10 min, the supernatants were collected and the analytes measured.

Test 2: Four fish from each gender were used for the whole-body concentration determination and another four were used for the tissue determinations. Livers, gonads and muscles were surgically dissected and weighed. The organs were then transferred to Lysing Matrix-D Tubes (MP Biomedicals, Solon, Ohio, USA) for tissue processing. The wholebody fish and tissue samples were homogenized using the FastPrep-24 Tissue and Cell Homogenizer as described above.

Analyte identification and quantitation were performed using an Acquity ultra performance liquid chromatography system (UPLC) coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA). UPLC separation was conducted on an Acquity BEH C18 column (2.1 mm x 100 mm; 1.7 µm; Waters). The mobile phases were LC-MS grade methanol and water. The flow rate was set at 0.4 mL/min, and the injection volume was 5 μL. The initial gradient conditions were 40% methanol for 1 min,
followed by a linear increase to 80% methanol over 5 min. Methanol was increased to 100% at 6.1 min, held for 2.0 min, and finally returned to the initial state to equilibrate for 2 min before the next injection. MS/MS acquisition was operated in negative-ion mode with multiple reaction monitoring (MRM). The capillary voltage was 2.9 kV. The source temperature and desolvation temperatures were 150 °C and 400 °C, respectively. Nitrogen gas (purity 99%) was used as the cone and desolvation gas at flow rates of 150 L/h and 1000 L/h, respectively. For each analyte, two transitions were selected for identification and the corresponding cone voltage and collision energy were optimized for maximum intensity.

To ensure that the samples could be accurately analyzed, an isotopic internal standard curve using deuterated BPAF at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 ng/mL were prepared for the determination of BPAF.
Vehicle:
not specified
Remarks:
stock solution prepared in ethanol but it is not clear from the publication if treatment solutions were prepared using ethanol as a co-solvent
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Not reported
- Controls: Dilution water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not reported
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): Not reported
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): All the fish were fed with freshly hatched Artemia nauplii (Fengnian Aquaculture Co., Ltd. Tianjin, China) twice and flake food (Tetra, Germany) once daily.
- Details of spiking (e.g. i) liquid test material (neat); ii) with a vehicle (corn or fish oil); or iii) using an organic solvent: Not reported
- Quantity of corn or fish oil vehicle, if used, per unit mass of fish food: n/a
- Chemical name of vehicle (organic solvent), if used: Not reported
- Method of mixing: Not reported
- Equilibration time: Not reported
- Method for removal of solvent, if used: Not reported
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Strain: AB
- Source: Department of Biological Sciences and Biotechnology, Tsinghua University (Beijing, China).
- Age at study initiation (mean and range, SD): 6 months
- Length at study initiation (length definition, mean, range and SD): Not reported
- Weight at study initiation (mean and range, SD): Not reported
- Weight at termination (mean and range, SD): Not reported
- Method of breeding: Not reported
- Lipid content at test initiation (mean and range, SD): Not reported
- Health status: Not reported
- Description of housing/holding area: The fish were cultured in a flow-through system with conditioned water at 27 ± 1 °C with a photoperiod of 14:10 h light/dark cycles. 10–20% of the
total fish water volume was changed with conditioned water on a daily basis. All the fish were fed with freshly hatched Artemia nauplii (Fengnian Aquaculture Co., Ltd. Tianjin, China) twice and flake food (Tetra, Germany) once daily. In order to ensure high water quality, food remains and debris were removed daily.
- Feeding during test
- Food type: Not reported
- Amount: Not reported
- Frequency: Not reported

ACCLIMATION
- Acclimation period: Not reported
- Acclimation conditions (same as test or not): Not reported
- Type and amount of food: Not reported
- Feeding frequency: Not reported
- Health during acclimation (any mortality observed): Not reported
Route of exposure:
aqueous
Test type:
semi-static
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
168 h
Total depuration duration:
24 h
Hardness:
Not reported
Test temperature:
27 ± 1 ºC
pH:
Not reported
Dissolved oxygen:
Not reported
TOC:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquarium
- Type (delete if not applicable):Not reported
- Material, size, headspace, fill volume: Glass, 15 L (18 cm x 30 cm x 30 cm)
- Aeration: Not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): 12 h

Test 1

- No. of organisms per vessel: 12 (6 males and 6 females)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: not reported

Test 2

- No. of organisms per vessel: 60 (30 males and 30 females approx.)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 0
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 75 g NaHCO3, 18 g sea salt and 8.4 g CaSO4 dissolved in 1000 L of deionised water.
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductance: Not reported
- Holding medium different from test medium: No
- Intervals of water quality measurement: Not reported
- Intervals of test medium replacement: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: Not reported
- Light intensity: Not reported
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: n/a
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): n/a
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: n/a
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: n/a

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 20 µg/L
- Results used to determine the conditions for the definitive study: stability of BPAF in the test system determined. Test solution renewals every 12 h would maintain test soltion concetrations within 10 % of initial value. Steady state in fish recorded after an uptake phase of 7 days- main test uptake phase would therefore reflect this.
Nominal and measured concentrations:
Nominal; Test 1- 1, 2, 5, 10 and 20 µg/L
Nominal; Test 2- 20 µg/L
Reference substance (positive control):
not required
Details on estimation of bioconcentration:
BASIS INFORMATION
- Measured/calculated logPow: 2.82
- Results from toxicokinetic study: Not reported
- Results from residue study: Not reported
- Monitoring data: Not reported

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFss determined only
- Result based on measured log Pow of: Not reported
- Result based on calculated log Pow of: Not reported

SPSS for Windows 13.0 Software was used to conduct one-way analysis of variance (ANOVA) to calculate statistical significance between each male and female groups.
Key result
Conc. / dose:
>= 1 - <= 20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
9 L/kg
Basis:
whole body w.w.
Time of plateau:
168 h
Calculation basis:
steady state
Remarks on result:
other: males, Test 1
Key result
Conc. / dose:
>= 1 - <= 20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
5.2 L/kg
Basis:
whole body w.w.
Time of plateau:
168 h
Calculation basis:
steady state
Remarks on result:
other: females, Test 1
Key result
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
9.8 L/kg
Basis:
whole body w.w.
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: males, Test 2
Key result
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
5.3 L/kg
Basis:
whole body w.w.
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: females, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
36.9 L/kg
Basis:
organ w.w.
Remarks:
liver
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: males, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
10.3 L/kg
Basis:
organ w.w.
Remarks:
liver
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: females, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
27.7 L/kg
Basis:
organ w.w.
Remarks:
gonads
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: males, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
10.2 L/kg
Basis:
organ w.w.
Remarks:
gonads
Time of plateau:
6 h
Calculation basis:
steady state
Remarks on result:
other: females, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
9.4 L/kg
Basis:
edible fraction
Remarks:
muscle
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other:
Remarks:
males, Test 2
Conc. / dose:
20 µg/L
Temp.:
>= 26 - <= 28 °C
Type:
BCF
Value:
4.6 L/kg
Basis:
edible fraction
Time of plateau:
72 h
Calculation basis:
steady state
Remarks on result:
other: female, Test 2
Key result
Elimination:
yes
Parameter:
other: BPAF concentrations in fish reduced by 80 %
Depuration time (DT):
24 h
Metabolites:
During the uptake phase, BPAF-G was the major BPAF metabolite and could be detected from the beginning of the accumulation phase. The appearance of BPAF-G at the start of the uptake phase indicated a rapid biotransformation of BPAF to BPAF-G in vivo.
Validity criteria fulfilled:
not specified
Conclusions:
The steady state BCF for Bisphenol-AF in whole fish tissue was 5.2 - 9.8 L/kg, indicating that the test item does not have the potential to bioaccumulate in zebrafish.
Bisphenol-AF concentrations in whole fish tissues rapidly dissipated after zebrafish were introduced to untreated dilution water (80 % reduction in test item concentraton in whole fish tissue after 24 h depuration).

Lipid content of fish was not measured so the BCF values cannot be corrected for lipid content- this ommision of data is unlikely to impact on the overall conclusion that BPAF will not bioaccumulate as the BCFss values are extremely low and well below the trigger value of 2000 L/Kg.
Executive summary:

The bioaccumulation potential of Bisphenol-AF (BFAP) to adult zebrafish (Danio rerio) was studied under semi-static conditions in accordance with OECD 305.  In an initial experiment 6-month old zebrafish were exposed to nominal concentrations of 1, 2, 5, 10 and 20 µg/L BPAF.  The test system was maintained at 27 ± 1 ºC for 168 h, the duration where a steady state in fish tissues was previosly determined. The 168 h BCFss (bioconcentration factor at a steady state) was 5.2 - 9.0 L/Kg for male and female fish. In an additional test, the 72 h BCFss was 5.3 - 9.8 L/Kg for whole fish and 10.2 - 36.9 L/Kg for organ tissue of male and female fish. At the end of a 24 h depuration phase, 80 % of whole fish BPAF concentrations had dissipated demonstrating that the test item was rapidly eliminated from the fish. The results conclude that BPAF would not bioaccumulate in zebrafish.

This accumulation study is classified as acceptable since it was conducted in accordance with OECD testing guidelines number 305 "Bioaccumulation in Fish: Aquatic and Dietary Exposure", although some of the validity criteria from the guidance document have not been reported i.e. chemical analysis of the water and water quality parameters.

Results Synopsis

Test Organism: Zebrafish (Danio rerio)

Test Type: Semi-static

BCFss (whole fish): 5.2 - 9.8 L/Kg

Description of key information

BCF = 52. - 9.8 L/kg; OECD 305; Shi et al. (2016)

Key value for chemical safety assessment

BCF (aquatic species):
9.8 L/kg ww

Additional information

The bioaccumulation potential of Bisphenol-AF (BFAP) to adult zebrafish (Danio rerio) was studied under semi-static conditions in accordance with OECD 305.  In an initial experiment 6-month old zebrafish were exposed to nominal concentrations of 1, 2, 5, 10 and 20 µg/L BPAF.  The test system was maintained at 27 ± 1 ºC for 168 h, the duration where a steady state in fish tissues was previosly determined. The 168 h BCFss (bioconcentration factor at a steady state) was 5.2 - 9.0 L/Kg for male and female fish. In an additional test, the 72 h BCFss was 5.3 - 9.8 L/Kg for whole fish and 10.2 - 36.9 L/Kg for organ tissue of male and female fish. At the end of a 24 h depuration phase, 80 % of whole fish BPAF concentrations had dissipated demonstrating that the test item was rapidly eliminated from the fish. The results conclude that BPAF is not considered bioaccumulative.