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REACH

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

EC number: 216-036-7 | CAS number: 1478-61-1

Life Cycle description
Uses advised against

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference 1

Endpoint:

long-term toxicity to fish, other

Remarks:

Fish Sexual Development Test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 234 Fish Sexual Development Test

Deviations:

yes

Remarks:

VTG concentrations were not determined, moreover the expression of the VTG gene through measurements of mRNA was used as an indicator of estrogen receptor stimulation

GLP compliance:

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: not reported
- Stability under test conditions: not reported
- Solubility and stability of the test substance in the solvent/vehicle: soluble in ethanol at 10 mg/L
- 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 create a stock solution at a concentration of 10 mg/L.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 10 mg/L
- 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

Analytical monitoring:

Details on sampling:

n/a

Vehicle:

yes

Remarks:

ethanol used to prepare stock solution

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions prepared from a 10 mg/L stock solution. Stock solution spiked into the appropriate test medium, depending on the phase of the test.
- Eluate: Egg water: distiled water with 60 mg/L sea salt) used until larvae had hatched (ca. 3 - 5 day post-fertilisation (dpf)). Embryo medium (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3) used until 40 dpf, after which time the test solutions were prepared in charcoal-filtered, dechlorinated, filtered tap water.
- Differential loading: 120 embryos incubated in 500 mL egg water. Surviving hatched larvae were incubated in 500 mL embryo water unti 14 dpf. On 14 dpf, about 50 larvae were incubated in 10 L of embryo medium.
- Controls: test media plus ethanol at the same rate as test vessels
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Strain: AB strain
- Source: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China
- Pre-exposure reproductive performance: Fish were cultured in a flow-through system with charcoal-dechlorinated filtered tap water at 27 ± 1 ºC with a photoperiod of 14:10 light:dark cycle. Adult fish were fed with freshly hatche Artemia nauplii twice and flake food once daily.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): not reported
- Method of collection of fertilised eggs: Fertilised embryos for the experiments were obtained from non-exposed adult fish and reared in egg water. Normal embryos were examined with a stereomicroscope. Those that had reached the blastula stage (30 % epiboly, ca. 4 hpf) were selected for the experiments.
- Subsequent handling of eggs: embryos transferred into 500 mL treatment solution after acceptance on to the study.

POST-HATCH FEEDING
- Start date: immediately from hatching
- Type/source of feed: From hatching to 14 days post fertilisation, fish were fed with Paramecium twice daily. From 14 dpf onwards, juveniles were fed Artemia nauplii twice daily.
- Amount given: not reported
- Frequency of feeding: twice daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

Total exposure duration:

120 d

Remarks on exposure duration:

After exposure the fecundity and fertilisation success of the zebra fish was investigated in untreated media.

Hardness:

not reported

Test temperature:

27 ± 1 ºC

pH:

not reported

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Nominal

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): not used
- Test vessel: 1 L glass beakers (up to 14 dpf) (18 cm diameter x 8 cm height), 15 L glass tank (up to 120 dpf) (18 x 30 x 30 cm) and 2 L fertilisation box used beyond 120 dpf to investgate fecundity.
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass beaker = 500 mL test solution, glass tank = 10 L, fertilisation box = 2 L
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): beaker solutions renewed daily up to 14 dpf thereafter, 50 % of tank contents were renewed daily.
- No. of fertilized eggs/embryos per vessel: 120 embryos per beaker up to 14 dpf. 50 larvae transferred to tanks on 14 dpf. To investigate fecundity, 4 males and 2 females were chosen from each tank.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 0
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Egg water = distilled water with 60 mg/L sea salt. Embryo medium = 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MGSO4, 4.2 mM NaHCO3
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Salinity: not reported
- Culture medium different from test medium: Not for embryo stage. Once hatched, larvae were transferred inot embryo medium.
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Embryo stage: hatch time, hatchability, survival rates. Larval stage: survival. Reproduction stage: Fecundity, fertilisation.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: no
- Results used to determine the conditions for the definitive study: n/a

POST-HATCH DETAILS
- Begin of post-hatch period: 3-5 days post fertilisation
- No. of hatched eggs (alevins)/treatment released to the test chamber: 120
- Release of alevins from incubation cups to test chamber on day no.: n/a

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30
- Removal of eggs to check the embryonic development on day no.: every 24 h until 7 days post fertilisation.

Reference substance (positive control):

Key result

Duration:

120 d

Dose descriptor:

NOEC

Effect conc.:

> 125 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Key result

Duration:

120 d

Dose descriptor:

NOEC

Effect conc.:

< 5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks:

Details on results:

Reproduction of F0 and F1 generations

In the F0 generation, no obvious difference on the hatch time, hatchability and survival rates between solvent control (SC) and exposure groups were observed during the 120 d exposure of BPAF. The fecundity of female was decreased from 182 ± 22 in SC to 167 ± 33 in the 125 µg/L exposure group, but the decrease was not statistically significant. However, a significant reduction in fertilisation success of spawn eggs was found in the 125 µg/L BPAF exposure group (49.1 ± 13.2 %, as compared to 75.6 ± 10.5 % in the control).

The malformation rates of the F1 generation 125 µg/L exposure group were significantly higher than SC group after 3 dpf. Because most malformed larvae died in the first few days, the survival rates of the 125 µg/L exposure group were significantly lower than SC group after 6 dpf. There was no significant difference in malformation rates in the 5 and 25 µg/L exposure groups compared with SC, but a significant decrease in hatchability rate was observed at 3 dpf in all exposure groups. The embryos that did not hatch within 6 dpf were dead.

Hormone concentrations in F0 generation

Exposure to BPAF caused statistically significant effects on steroid hormone in fish. In males, the concentrations of plasma E2 (17b-estradiol) were significantly increased by 226 % and 277 % in 25 and 125 µg/L exposure groups compared with the SC. The concentrations of plasma T (testosterone) were significantly reduced by 57 % and 67 % in 25 and 125 µg/L exposure groups compared with the SC. These changes in steroid hormone concentrations led to significant increases of E2/T ratios at 25 and 125 µg/L BPAF groups in male fish. In females, E2 levels were significantly increased by 126 % in 125 µg/L group compared with SC but T levels were unchanged in any of BPAF exposure groups. The ratio of E2/T was significantly increased in the 125 µg/L exposure group in female fish.

Gene expression in F0 generation

In the liver of male fish, vtg1 was significantly increased in 25 and 125 µg/L exposure groups compared with SC. However, the expression of vtg1 remained unchanged in female fish. In the brains, expressions of gnrh2, fshb, lhb, and cyp19b in male were significantly increased in the 125 µg/L exposure compared with SC. However, no differences were found in the brain tissues obtained from female fish. In the gonads, expressions of fshr, cyp19a and cyp11a1 were significantly increased and star with cyp17 was significantly decreased in testis of the 125 µg/L exposure group compared with SC. In ovaries, compared with SC, an increase of fshr and a decrease of star were found in the 125 µg/L exposure group.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

All data were analyzed using SPSS for Windows 13.0 Software and presented as mean ± standard error of the mean (SEM). Homogeneity of variances was analyzed by Levene’s test. Normality of the data was tested using the Kolmogorov–Smirnov test. Oneway analysis of variance (ANOVA) was applied to calculate statistical significance. If a statistically significant difference (P < 0.05) (Han et al., 2011) was revealed by the ANOVA test, Dunett’s test was used to identify differences between exposure groups and the SC.

Table 1: Malformation, hatchability and survival rates of F1 generation following parent (F0 generation) exposure to BPAF and ethanol (solvent control)

Time

(dpf)

Malformation Rate (%)

Hatchability (%)

Survivability (%)

5 µg/L

25 µg/L

125 µg/L

5 µg/L

25 µg/L

125 µg/L

5 µg/L

25 µg/L

125 µg/L

2.4 ± 2.1

3.6 ± 2.2

1.9 ± 2.3

3.7 ± 2.3

0.0

96.4 ± 2.3

94.3 ± 4.0

93.9 ± 4.7

96.0 ± 27

1.9 ± 1.3

3.4 ± 2.1

2.6 ± 3.2

2.3 ± 1.9

0.0

95.8 ± 3.2

93.9 ± 3.2

92.6 ± 5.8

94.8 ± 3.8

11.6 ± 5.7

11.8 ± 5.6

13.1 ± 9.8

60.7 ± 24.6*

33.5 ± 23.4

13.3 ± 12.8*

6.1 ± 4.3*

13.7 ± 12.6

94.4 ± 4.0

91.7 ± 4.5

92.6 ± 6.2

93.9 ± 6.1

9.6 ± 8.9

8.3 ± 6.7

12.5 ± 7.6

58.1 ± 22.3*

81.8 ± 15.7

78.4 ± 13.5

77.4 ± 15.6

79.3 ± 14.6

94.4 ± 4.1

91.7 ± 4.7

92.6 ± 6.3

93.9 ± 6.2

9.6 ± 8.7

7.7 ± 5.5

12.5 ± 7.3

58.0 ± 22.2*

84.5 ± 14.8

85.2 ± 10.2

84.0 ± 12.4

84.5 ± 10.9

94.4 ± 4.2

91.1 ± 6.8

92.6 ± 6.9

93.6 ± 6.4

9.5 ± 7.2

7.5 ± 5.3

12.4 ± 8.9

58.0 ± 21.9*

90.5 ± 8.6

89.8 ± 9.8

88.3 ± 9.5

90.9 ± 7.4

94.2 ± 6.7

90.9 ± 8.0

92.5 ± 7.3

73.6 ± 12.8*

3.2 ± 2.1

6.0 ± 5.1

6.5 ± 5.7

50.9 ± 20.1*

99.2 ± 20.0

90.5 ± 6.7

92.2 ± 6.8

93.8 ± 5.0

92.5 ± 7.8

88.1 ± 7.2

89.4 ± 8.2

45.9 ± 20.6*

Table 2: Expression of selected genes along the HPG axis and liver of zebrafish (F0 generation) after exposre to BPAF and ethanol (solvent control)

Tissue	Gene name	Male				Female
Tissue	Gene name	SC	5 µg/L	25 µg/L	125 µg/L	SC	5 µg/L	25 µg/L	125 µg/L
Brain	gnrh2	1.13 ± 0.13	1.81 ± 0.36	1.04 ± 0.52	2.01 ± 0.78*	1.03 ± 0.36	0.98 ± 0.57	1.14 ± 0.23	1.17 ± 0.47
	fshb	1.04 ± 0.12	1.26 ± 0.23	0.84 ± 0.37	3.79 ± 1.35*	1.06 ± 0.12	1.15 ± 0.21	1.27 ± 0.23	1.50 ± 0.22
	lhb	1.02 ± 0.09	1.06 ± 0.25	1.87 ± 0.49	2.51 ± 1.07	1.02 ± 0.37	1.20 ± 0.33	0.88 ± 0.36	1.25 ± 0.47
	cyp19b	1.03 ± 0.14	1.31 ± 0.25	0.77 ± 0.33	1.59 ± 0.65*	1.00 ± 0.12	0.73 ± 0.32	1.19 ± 0.33	0.93 ± 0.42
	ERa	1.05 ± 0.07	1.03 ± 0.21	1.11 ± 0.32	0.97 ± 0.23	1.22 ± 0.34	1.23 ± 0.25	1.42 ± 0.69	2.26 ± 1.12
	ERb	0.97 ± 0.15	0.81 ± 0.21	1.08 ± 0.23	0.85 ± 0.23	1.08 ± 0.20	1.44 ± 0.35	0.85 ± 0.36	1.84 ± 0.95

Gonad	fshr	1.05 ± 0.16	0.83 ± 0.23	1.07 ± 0.59	2.38 ± 1.30*	1.01 ± 0.32	1.1 ± 0.07	1.21 ± 0.54	1.90 ± 0.79*
	lhr	1.01 ± 0.18	0.81 ± 0.32	0.60 ± 0.39	0.85 ± 0.42	1.00 ± 0.23	1.00 ± 0.35	1.05 ± 0.53	1.03 ± 0.45
	str	1.09 ± 0.21	0.97 ± 0.25	0.84 ± 0.21	0.45 ± 0.13*	1.07 ± 0.12	1.01 ± 0.32	1.00 ± 0.54	0.55 ± 0.23*
	cyp11a1	1.00 ± 0.06	0.79 ± 0.23	1.88 ± 0.79	2.32 ± 1.09*	1.04 ± 0.23	1.11 ± 0.45	0.90 ± 0.23	0.66 ± 0.31
	cyp17	1.06 ± 0.21	0.97 ± 0.36	0.76 ± 0.25	0.66 ± 0.12*	1.01 ± 0.31	0.97 ± 0.39	1.64 ± 0.95	1.49 ± 0.58
	cyap19a	1.19 ± 0.13	0.99 ± 0.59	1.27 ± 0.13	2.07 ± 0.62*	1.04 ± 0.12	1.09 ± 0.35	0.78 ± 0.25	1.08 ± 0.54
	hsd3b	1.00 ± 0.08	0.96 ± 0.14	1.45 ± 0.69	1.16 ± 0.65	1.00 ± 0.09	0.96 ± 0.24	0.86 ± 0.23	1.24 ± 0.36
	hsd17b	1.06 ± 0.13	0.72 ± 0.26	0.88 ± 012	0.91 ± 0.08	1.04 ± 0.12	0.80 ± 0.46	0.98 ± 0.66	0.85 ± 0.25
	hmgra	1.07 ± 0.22	1.19 ± 0.53	1.28 ± 0.55	1.58 ± 0.54	1.11 ± 0.28	0.85 ± 0.42	0.71 ± 0.37	0.89 ± 0.32
	hmgrb	1.00 ± 0.32	1.06 ± 0.53	1.25 ± 0.53	1.13 ± 0.66	1.01 ± 0.22	0.97 ± 0.25	0.89 ± 0.21	0.75 ± 0.33

Liver	vtg1	1.05 ± 0.40	21 ± 20	470 ± 172*	1214 ± 208*	1.00 ± 0.14	0.90 ± 0.11	1.12 ± 0.23	1.27 ± 0.21

The results are shown as mean ± standard error of mean (SEM) (n = 6). Asterisk indicates significant difference from solvent control (SC) (P < 0.05).

Validity criteria fulfilled:

not specified

Conclusions:

For the F0 generation, no obvious difference on the hatch time, hatchability and survival rates between SC and exposure groups were observed. The NOEC for mortality for the F0 generation could therfore be described as greater than or equal to 125 µg/L.

However, breeding success of the F1 generation was significantly reduced in the 125 µg/L concentration group where a decrease in testosterone (males) and an increase in estradiol (males and females) in the precedding generation of fish resulted in adverse effects on reproduction. The expression of selected genes along the HPG axis and liver (critical in the role of reproduction) were also adversly affected, as a consequence of the hormone imbalance.

Bisphenol AF has endocrine disrupting potential at concentrations at 5 µg/L, indicated by a delay in hatching of F1 generation offspring (the most sensitive parameter). The NOEC for adverse effects for the F1 generation could therfore be described as being < 5 µg/L.

Executive summary:

The effect of Bisphenol AF on the toxicity and reproductivity of the zebrafish Danio rerio was investigated at 3 concentrations in addition to an ethanol solvent control. The hatchability and survivability of fertilised embryos and hatched larvae maintained at the 3 concentrations was observed over a period of 120 days after which time the levels of plasma hormones and the expression of select genes along the hypothalamus-pituitary-gonad (HPG) axis and liver were measured. Select F0 generation adults were also bred and the fertilisation, hatchability, malformation and survivability of eggs in the F1 generation observed. A combination of the physiological and biochemical observations would describe the endocrine disrupting potenital of Bisphenol AF.

The present study indicates that exposure to BPAF did not impact on the hatchability and survival rates of the F0 generation of zebrafish however it could modulate the steroid hormonal balance of individuals and impact the development of offspring. The significant decreases in egg fertilization of offspring may indicate the possibility of sperm deterioration of parent following exposure of BPAF. The higher occurrence of malformation and lower survival rate in the offspring of exposure group suggest a possibility of maternal transfer of BPAF that could increase the prevalence of adverse health signs in the offspring. The hatching delay observed in 5 µg/L BPAF indicated that parental exposure to environmentally relevant concentration of BPAF would result in delayed hatching of the offspring.

The NOEC for mortality could be described as > 125 µg/L where survivability at the top tested concentration was not significantly reduced in comparison to the control group. However, the NOEC for sublethal effects could be described as being < 5 µg/L where hatchability of the F1 generation was significantly reduced at the lowest tested concetration in comparison to the control, as a result of hormone imbalance. The results of this test suggest that Bisphenol AF could have endocrine disrupting potential.

Applicant's interpretation:

In this study, similar to an OECD 210 (2013), the toxicity in the early life stages of fish was assessed, further though the authors also conducted an assessment on the impact of the F1 generation.

Fertilized embryos for the experiments were obtained from non-exposed adult fish. Normal embryos were examined with a stereomicroscope (SMZ1000, Nikon, Japan). Those that had reached the blastula stage (30% epiboly, about 4 hpf) were selected for the experiments. Four exposure conditions with three replicates each were set up. Approximately 120 embryos were used per concentration, or in the solvent control. The dead embryos were recorded and removed as soon as possible. After hatch (about 3–5 day-post-fertilization (dpf)), the larvae were transferred into 1-L glass beaker containing 500 mL of embryo medium with the same concentrations of test item and fed with Paramecium twice daily under semi-static conditions (complete renewal of solutions after 24 h) until 14 dpf. At 14 dpf, about 50 normal larvae were randomly chosen from each beaker and distributed into a 15-L glass tank (18 cm 30 cm 30 cm) containing 10 L embryo medium with the same concentrations of test item and begun to feed with Artemia nauplii twice daily. The larvae were exposed to test item in a semi-static system and the exposure medium was half-renewed every day. During the experimental period, residual food and feces in the test tanks were removed and the test tanks were cleaned once a week. After 40 dpf, all of the fish were fed with Artemia nauplii and the charcoal-dechlorinated filtered tap water replaced the embryo medium until the end of the exposure (120 dpf). The hatch time, hatchability and survival rates of F0 generation during the exposure were recorded. At 120 dpf the genders of the fish were phenotypically determined and separated. Four males and two females were randomly sampled from each glass tank, and two males were paired with one female fish in a 2-L fertilization box without any solvent or test item. The total number of eggs produced by each female in the seven consecutive days was summed as fecundity of each female. Thirty spawned eggs for the first time spawn from each female were randomly collected from the fertilization box and they examined under a dissecting microscope for cell cleavage to verify fertilization. Fertilized embryos were reared in sterile Petri dishes with the egg water at 27 ± 1 °C without any solvent or test item, as described above. The number of hatched, dead and malformed embryo/larvae was recorded every 24 h until 7 dpf. The malformation and death of embryo were determined according to the OECD guideline 236 (2013). The larva without heartbeat was determined as dead. The fertilization rate was calculated as fertilized embryo/all selected eggs. Hormone and gene expression measures were also taken. However, under the current regulatory frameworks these endpoints are not usable for hazard assessment purposes.

For the validity criteria the temperature did not differ by more than 1.5 °C (actual: 27 ± 1 °C), the dissolved oxygen content was not confirmed but control fish showed no obvious signs of ill health, and the test concentrations were not measured. However even though the study only reports nominal concentrations, the chemical has been shown to stable in aquatic systems i.e. there is sufficient evidence within the literature to suggest that 1) test item is stable in environmental matrices, 2) the chemical is sufficiently soluble at the tested level, and 3) the test item will not shift pH or have a high oxygen demand. For example Tisler et al. 2016 verified during a chronic Daphnia magna test that the test item was stable (i.e. within 90 % of nominal dosed concentrations) after 3 days in Daphnia medium (Elendt M4). In an algae study conducted by DuPont 2007, analytical measures showed that the test item was stable (between 80-120 % of nominal) for the duration of the test (72 hours). The medium used here is much less complex and similar stability would be expected, furthermore the water is replenished frequently and so the time-frame for stability also applies. Feeding frequency compiled with previous suggestions for the fish, although a different food source was used, fish remained healthy.

For hatch time, hatchability and survival rates between SC and exposure groups were observed during the 120 d exposure (i.e. similar to the OECD 210 guideline). Therefore the LC50 and NOEC are determined to be > 125 µg/L in support of observations made within the literature (Yang et al., 2014). The duration is 4 times longer than that required by the OECD 210 guideline and provides sufficient evidence that there is no biological impact on this generation at 125 µg/L.

There were significant malformations and impacts on survival in the F1 generation at 125 µg/L. No other significant effects were consistently observed in the other concentrations. However it is not yet understood how such data should be used in the context of a regulatory framework for hazard assessment.

The key endpoint from the study is an F0 NOEC of > 125 µg/L.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 234 (2011)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Principles of method if other than guideline:

- Principle of test:

Hatchability and survivability of fertilised Danio rerio embryos maintained in 3 concentrations of Bisphenol AF (BPAF), in addition to a solvent control was investigated. In addition, haematological analysis was conducted on two-month old zebra fish maintained at 28 ºC in three concentrations of BPAF for 21 days.

- Short description of test conditions:

Embryo development/ survivability test

Zebrafish embryos were collected and rinsed with an embryo medium (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3). Healthy embryos were selected with a dissecting microscope and incubated in dishes at 28 ± 0.5 ºC until chemical treatment. The embryos were exposed in 4 mL glass vials to BPAF at different concentrations according to the preliminary study (0.5, 1.0 and 1.5 mg/L). Chemicals concentrations in exposure water were analyzed by liquid chromatography/mass spectrometry (LC/MS). A vehicle and a nonvehicle control were included to confirm that DMSO concentration used had no effect on zebrafish development. Studies were conducted with 30 embryos per dose and all studies were repeated at least three times. Developmental lesions, including edema and hemorrhage, as well as death and date of hatching, were recorded daily.

Endocrine disruption potential test - in vivo

Two-month-old male zebrafish was cultured in recirculating aquarium tanks at 28 ± 0.5 ºC. The fish was maintained in a 14:10 light/dark cycle and fed twice daily with fresh Artemia nauplii. Exposure studies were performed in 10 L glass aquaria. Ten males selected randomly from acclimatized fish were put into each aquaria and triplicate aquaria were used for each exposure concentration (as above). The stock solutions were added into the exposure tanks using a dilution apparatus. Static conditions were applied for the exposures and the total volume of solution was changed every day. The exposure duration was 21 d and the fish were fed daily. The blood of zebrafish was collected in freshly heparinized microhematocrit tubes by severing the caudal peduncle, and centrifuged at 11000 rpm for 10 min. After centrifugation, serum volumes (2–10 µL) were obtained from individual adult zebrafish, and subsequently diluted in phosphate-buffered saline (PBS) and frozen at 80 ºC until analysis. Vtg was purified from serum of E2-induced zebrafish and used as a standard after identification and quantification. Serum vtg levels were determined using a comparative enzyme-linked immunosorbent assay (ELISA).

Endocrine disruption potential test- in vitro

The relative estrogenic activity was determined by MVLN assay. This cell line was stably transfected with the luciferase reporter gene and estrogen-responsive element, where ER agonists can induce the production of luciferase. Briefly, 60 interior wells of a 96-well culture plate were each seeded with 250 µL cell suspension at a density of about 5x10E4 cells per well. Cells were cultured under aseptic conditions in a humidified CO2 incubator at 37 ºC and 5% CO2. The cells were starved in steroid-free medium for 48 h before exposure. A concentration range of 0.1 pM to 10 nM E2 was used as a positive control. Luciferase activity was measured using the LucLite kit according to the manufacturer’s protocol. Luminescence was read by Microplate Reader (Varioskan Flash) after cells were exposed to test compound for 48 h, and total protein content was simultaneously measured by the Bradford assay to normalize luminescent units. The maximal luciferase activity induction of E2 was set as 100%, and the responses of other samples were converted to a percentage of the maximum level. Four replicates were used in each experiment.

- Parameters analysed / observed:

Embryo survivability - hatchability (hatching time and hatching rate) and mortality/survivability
Endocrine disruption potential test - in vivo - VTG concentration
Endocrine disruption potential test - in vitro - induced luciferase activity

GLP compliance:

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: stock solution stored in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solid dissolved in DMSO
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: not reported
- Final preparation of a solid: Dissolved in DMSO

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

OTHER SPECIFICS: n/a

Analytical monitoring:

yes

Remarks:

LC/MS sampling, conditions and results reported in a supplementary paper (Song et al., 2014)

Details on sampling:

Not reported

Vehicle:

yes

Remarks:

Dimethylsulfoxide (DMSO)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Not reported
- Eluate:

Embryo development test: Embryo medium 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3

In vivo endocrine disruption potential test: not reported

- Differential loading:

Embryo development test: 30 embryos per dose (4 mL)

In vivo endocrine disruption potential test: 10 males per dose (10 L tank)

- Controls: Yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not reported
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not reported

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Embryo Development Test

TEST ORGANISM
- Common name: Danio rerio
- Strain: not reported
- Source: Reported in supplementary papers
- Pre-exposure reproductive performance: Not reported

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Reported in a supplementary publication (Jin et al., 2009; Li et al., 2011; Chan and Chan, 2012)
- Method of collection of fertilised eggs: Reported in a supplementary publication (Jin et al., 2009; Li et al., 2011; Chan and Chan, 2012)
- Subsequent handling of eggs: Rinsed in embryo medium. Healthy embryos were selected with a dissecting microscope and incubated in dishes at 28 ± 0.5 ºC until chemical treatment. Studies were conducted with 30 embryos per dose and all studies were repeated at least three times.

Reproduction Test

Two-month-old male zebrafish was cultured in recirculating aquarium tanks at 28 ± 0.5 ºC. The fish was maintained in a 14:10 light/dark cycle and fed twice daily with fresh Artemia nauplii. Exposure studies were performed in 10 L glass aquaria. Ten males selected randomly from acclimatized fish were put into each aquaria and triplicate aquaria were used for each exposure concentration.

POST-HATCH FEEDING
- Start date: Not reported
- Type/source of feed: Artemia nauplii
- Amount given: Fed twice daily, feeding rate not reported.
- Frequency of feeding: Twice daily.

Test type:

other: Embryo test: Not reported, Reproduction test: Semi-static, with daily changes.

Water media type:

freshwater

Limit test:

Remarks on exposure duration:

Embryo test: 144 hours post fertilisation, Reproduction test: 21 days

Hardness:

Not reported

Test temperature:

28 ± 0.5 ºC

pH:

Not reported

Dissolved oxygen:

Not reported

Salinity:

n/a

Conductivity:

Not reported

Nominal and measured concentrations:

Embryo test (nominal): 0.5, 1.0, 1.5, 2.0 mg/L and Hormone test (nominal): 0.5, 1.0, 1.5 mg/L
Measured: Not reported

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): n/a
- Test vessel: Embryo test: 4 mL glass vials, Hormone test: 10 L glass tank
- Type (delete if not applicable): Not reported
- Material, size, headspace, fill volume: Glass, fill volumes not reported.
- Aeration: Not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- No. of fertilized eggs/embryos per vessel: Embryo test: 30 embryos per concentration, Hormone test: n/a
- No. of adult males per vessel: Embryo test: n/a, Hormone test: 10 males per concentration
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Embryo medium (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3)
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Salinity: n/a
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 14:10 Light:Dark
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Embryo test: Hatching rate and mortality, Hormone test: Mortality, concetration of VTG, relative estrogenic activity (derived from MVLN)

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Yes

POST-HATCH DETAILS
- Begin of post-hatch period: n/a - embryo test concluded at 144 hour post fertilisation
- No. of hatched eggs (alevins)/treatment released to the test chamber: n/a - embryo test concluded at 144 hour post fertilisation
- Release of alevins from incubation cups to test chamber on day no.: n/a

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30 per dose
- Removal of eggs to check the embryonic development on day no.: Occurence of lesions in embryos was investigated daily

Reference substance (positive control):

yes

Remarks:

E2 (estradiol) used as a positive control (induction of vtg)

Key result

Duration:

144 h

Dose descriptor:

LC50

Effect conc.:

1.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

embryos

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hormone imbalance

Remarks:

increase in vtg induction in male zebrafish is an indication of estrogen receptor stimulation.

Key result

Duration:

144 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

larval development

Key result

Duration:

144 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

embryo / larvae

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: Embryo test: 100 % embryo mortality at 2.0 mg/L at 144 hours post fertilisation. Lower test concentrations did not impact on mortality by 144 hours post fertilisation. Hormone test: No significant increase in mortality was observed vs controls at all test concentrations.
- Days to hatch or time to release of young: Embryo test: test concentrations did not significantly impact on hatching time (gererally, all hatched within 48 - 72 h at 0.5, 1.0 and 1.5 mg/L)
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Embryo test: test concentrations did not significantly impact on hatching rate (generally > 90 % by 90 hpf)
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): not reported
- Observations on body length and weight of young and/or exposed parents at one or more time periods: not reported
- Number of healthy fish at end of test: Hormone test: test concentrations did not significantly impact on fish mortality after 21 days
- Type of and number with morphological abnormalities: Embryo test: significant increase in pericardial oedema (37.8 % of individuals vs. control value of 4.4 %) in embryos treated at 1.5 mg/L
- Type of and number with behavioural abnormalities: not reported
- Type and number of developmental/reproductive effects: Hormone test: vtg concentrations for 0.5, 1.0 and 1.5 mg/L were 932.4 ±24.8, 2236.2 ± 149.5 and 5292.3 ± 248.6 µg/mL, respectively. As a comparison, significant (p < 0.001) vtg induction was observed at a concentration of 5 µg/L estradiol (E2).
- Detailed data on spawning, egg numbers, fertility, and fecundity: Not conducted
- Type and magnitude of hormonal changes: Hormone test: Significant increase (p<0.001) in vtg concentrations observed in fish treated at all concentrations of BPAF. Relative estrogenic activity was noted with a maximal value of 72.3 % at 5 µM in the MVLN assay. Significant cytotoxity was observed at 10 µM BPAF in the MVLN assay.
- Other biological observations: n/a
- Effect concentrations exceeding solubility of substance in test medium: not reported
- Incidents in the course of the test which might have influenced the results: n/a

Results with reference substance (positive control):

The male zebrafish was highly responsive towards E2 treatment and a significant (p < 0.001) vtg induction was observed at a concentration of 5 µg/L.

Reported statistics and error estimates:

SPSS statistical software (Version 13.0) and Sigma Plot 10.0 were used for statistical analysis. The significant differences between control and treated groups were determined using a one-way analysis of variance and Tukey’s multiple range test. Differences were statistically significant if p < 0.05.

Embryo Test

The embryos of control group developed normally in embryo medium, and almost all embryos were hatched between 48 and 72 h. The exposure of BPAF (1.0 and 1.5 mg/L) slightly delayed the occurrence of hatching, but this finding was not statistically significant. All embryos/larvae died when exposed to 2 mg/L BPAF for 144 h however, lower exposure concentrations had no impact on embryos/larvae mortality in comparison to the control. The 144 h LC₅₀ value for BPAF for zebrafish larvae was 1.75 mg/L. The 144 h NOEC value for BPAF for zebrafish larvae was 1.0 mg/L, where statistically significant increases in pericardial oedema were observed at 1.5 mg/L.

Hormone Test

During 21 d of exposure to BPAF, no increase in mortality was observed compared to the control at all test conditions (0.5, 1.0 and 1.5 mg/L). The results of the vtg analysis in male zebrafish following 21 d exposure period to BPAF indicated a statistically significant (p<0.001) increase in vtg levels at all three concentrations tested compared to the controls. MVLN assay results were in accordance with vtg analysis results where relative estrogenic activity was elevated at all four concentrations (0.5, 1.0, 5.0 and 10.0 µM) in comparison to the controls. Significant cytotoxicity was also observed at the highest tested concentration. The NOEC could therefore be described as being < 0.5 mg/L in adult zebrafish.

Validity criteria fulfilled:

not specified

Conclusions:

The NOEC for zebrafish was not determinable in this study as significantly increased (p<0.001) vtg induction was observed in the lowest tested concentration (0.5 mg/L) in adult fish maintained for 21 days, the most sensitive parameter measured.

Executive summary:

The effect of Bisphenol AF on zebrafish was investigated in both post fertilised embryos and adult males for 144 hours post fertilisation and 21 days, respectivley. Embryos and adult fish were maintained at 28 ± 0.5 ºC.

In the embryo test, fertilised embryos were exposed to Bisphenol AF at four concentrations; 0.5, 1.0, 1.5 and 2.0 mg/L for up to 144 hours post fertilisation. During the test, daily observations were made for mortality, hatchability and sublethal effects, namely lesion development. The results showed that mortality in the three lowest-tested concentrations was not statistically different to the control group, however 100 % mortality was observed at 2.0 mg/L. The resulting LC₅₀ and NOEC values for mortality in embryos were determined to be 1.75 and 1.5 mg/L, respectively. Embryos were observed daily under a dissection microscope for signs of lesion development. It was noted that there was a statistically significant increase in the number of pericardial oedema present in the 1.5 mg/L group in comparison to the controls. The NOEC for sublethal effects in embryos could therefore be described as 1.0 mg/L, under the conditions of this test.

In the adult hormone test, no significant mortalities were observed at any of the three test concentrations (0.5, 1.0 and 1.5 mg/L), in comparison to the control group, after 21 days of exposure. The NOEC for adult fish mortality could be described as greater than or equal to 1.5 mg/L. Blood was collected from each fish after 21 day of exposure by severing the caudal peduncle. Concentrations of vtg in blood samples was determined using an ELISA assay. Vtg levels were significantly (p<0.001) increased at all BPAF tested concentrations as a result of estrogen (E2, estradiol) receptor stimulation. Results from a MVLN assay were in accordance with the results of the vtg assay where concentrations of BPAF at 5 µM could increase relative estrogen activity by 72.3 %. The NOEC for sublethal effects on adult zebrafish could not be established as significant effects were observed at the lowest tested concentration in the vtg assay.

Applicant's interpretation:

This was conducted using similar methods to those described in the OECD guidelines 234 (2011) and 236 (2013). However, the recommended duration for the tests was not met for the OECD 234 test (21-d test compared to guideline requirements of 60 day post fertilisation (OECD 234, 2011)). Further the OECD 234 is not a standard test listed for REACH endpoint fulfilment for long-term toxicity to fish (REACH legislation Annex IX). However, the study further supports that effects on length, weight, hatching success and survival/mortality are seen above 1 mg/L as seen in the study by Yang et al. (2014).

Zebrafish embryos were collected and rinsed with mineral medium. Only healthy embryos were selected for the test. It is unclear when the embryos were fertilised, but all measures are referred to as hours post fertilisation. A vehicle and non-vehicle control were included to confirm that the used level of DMSO was not detrimental to fish health. In a preliminary study following similar guidelines as the OECD 236 fish embryos were exposed in 4 mL glass vials to a range of concentrations between 0.5 and 10 mg/L for 144 hours. Studies were conducted with 30 embryos per dose and all studies were repeated at least three times. Developmental lesions, including oedema and hemorrhage, as well as death and date of hatching, were recorded daily. The LC50 was determined to 1.75 mg/L and the NOEC was 1.0 mg/L. Although 10 % mortality was observed within the 1 mg/L group this was not seen as biologically significant and still falls within the possible expected mortality of healthy unexposed populations which is further highlighted by the validity criteria which allow such mortality rates within the controls. Hatching rate was not significantly effected in all surviving fish. Developmental anomalies were detected in fish exposed to the test item at 1.5 mg/L but the NOEC for abnormalities (seen as oedema) was 1.0 mg/L. At 96 hours, there were no mortalities at any concentration and no effects on hatching rates.

For the part of the study which was similar to the OECD 234 guideline fish around 2 months of age were selected and exposed to 0.5, 1 and 1.5 mg/L of the test item. The fish were exposed for 21 days. Survival and VTG was measured at the end of the exposure period. No exposure level had an impact on the survival of fish. Vitallogenin was significantly higher at all tested concentrations. The test should be conducted on embryos and that move through to hatching, meaning it is not possible to apply some of the validity criteria. Due to this and the fact that the test is not used as a standard test for endpoint fulfilment it is not possible to use these endpoints in a meaningful risk and hazard assessment. However, as aforementioned, the results on survival further support an LC50 of above 1 mg/L.

The study only reports nominal concentrations, however concentrations were checked via LC/MS, but not results were presented. It was noted that the test concentrations (including DMSO) were homogenous in the test replicates during the study. There is sufficient evidence within the literature to suggest that 1) the test item is stable in environmental matrices, 2) the chemical is sufficiently soluble at the tested level, and 3) test item will not shift pH or have a high oxygen demand. For example Tisler et al. 2016 verified during a chronic Daphnia magna test that the test item was stable (i.e. within 90 % of nominal dosed concentrations) after 3 days in Daphnia medium (Elendt M4). The medium used here is much less complex and similar stability would be expected. Further, DMSO appears to be an appropriate solvent in all fish testing with high rates of hatching success and survival, and minimal noting of malformations or ill health in Song et al. (2014), Tang et al. (2015) and Yang et al. (2014), which all used DMSO as a solvent. Song et al. (2014) also included a DMSO and medium control to show that no significant effect was caused by the use of the solvent. An algae study conducted by DuPont (2007) and the chronic Daphnia magna study conducted by Tisler et al. (2016) provide further evidence that water quality parameters will not be greatly affected by these concentrations of the test item.

For validity criteria according to OECD 236 (2013) although temperature was 28 ± 0.5 °C, it was seen as acceptable for this species, it was also noted that the embryos of control group developed normally in embryo medium, and almost all embryos were hatched between 48 and 72 h likely complying with hatchability requirements of => 80 %. However, the survival in both controls, fertilisation success and positive control results on the embryos is unknown. Sufficient embryos were used per concentration and the test was repeated 3 times, giving some confidence in the presented results.

The study also shows that VTG can be affected with a NOEC of < 0.5 mg/L. The study shows survival of embryos and juvenile fish is unaffected at 1.5 mg/L. The 144 hour post fertilisation LC50 of 1.75 mg/L and a 144 hpf NOEC of 1.5 mg/L. In accordance with the guideline durations of 96-hours there were no observed significant mortalities and no effects on hatching rates at any exposure concentration.

Reference 3

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

not specified

Remarks:

it cannot be determined if the validity criteria of the guideline are met (dissolved O2)

GLP compliance:

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: stored away from light (stock solution)
- Stability under test conditions: not reported
- Solubility and stability of the test substance in the solvent/vehicle: soluble at 1 and 4 mg/L in DMSO (dimethylsulfoxide)
- 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: stock solutions prepared at 1 and 4 mg/L in DMSO (dimethylsulfoxide)
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 1 and 4 mg/L stock solutions prepared
- 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

Analytical monitoring:

Details on sampling:

n/a

Vehicle:

yes

Remarks:

DMSO (dimethylsulfoxide)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: BPAF was dissolved in dimethyl sulfoxide (DMSO) to form stock solutions (1 and
4 mg/L) which were stored away from light.
- Eluate: not reported
- Differential loading: not reported
- Controls: yes, 0.1 % DMSO
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 %
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish (Danio rerio)
- Strain: not reported
- Source: not reported
- Age at study initiation (mean and range, SD): 2 months old
- Weight at study initiation (mean and range, SD): females = 0.28 ± 0.02 g, males = 0.26 ± 0.02 g
- Method of breeding: not reported

FEEDING DURING TEST
- Food type: fresh Artemia nauplii
- Amount: not reported
- Frequency: twice daily

Test type:

semi-static

Water media type:

not specified

Limit test:

Total exposure duration:

28 d

Post exposure observation period:

n/a

Hardness:

not reported

Test temperature:

28 ± 0.5 ºC

pH:

not reported

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

nominal; 0.05, 0.25 and 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 6 L glass aquaria
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: not reported
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): 50 % of test solution replaced daily
- No. of organisms per vessel: 9
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): n/a (vehicle control only)
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Salinity: not reported
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : body weight and length were measured prior to test initiation and at Day 28. Histology, hormone and gene expression measurements also conducted on Day 28, after completion of the incubation phase.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: no
- Results used to determine the conditions for the definitive study: n/a

Reference substance (positive control):

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

male and female

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Fish weight and length increases (Day 0 vs. Day 28) (mean values): see Table 1
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): not reported
- Type of and number with morphological abnormalities:

Histology

The hepatocytes of male fish exposed to 1 mg/L BPAF were swollen and irregularly shaped, in contrast to the compact and polygonal-shaped hepatocytes in the control fish. In addition, it was noted that vacuolization in the liver in the groups exposed to 1 mg/L BPAF. However, BPAF treatment did not result in any hepatic damage in any female fish. Exposure to BPAF did not cause any obvious alterations in the gills and intestines of both sexes compared with the control. In males, germ cells were observed in all stages of spermatogenesis. Acellular areas in the testis of fish exposed to 1 mg/L BPAF was observed, suggesting a reduction of spermatids. In contrast to the control fish, the majority of cells from BPAF-treated females were in stage I, although a small number of cells were at stage IV. Exposure to 0.25 and 1 mg/L BPAF resulted in a significantly higher proportion of stage I cells and caused significantly lower proportion of stage IV cells relative to the control fish, suggesting that BPAF inhibits ovarian maturation.

Hormones

In male fish, the T levels in the whole-body homogenates were reduced in a dose-dependent manner. However, the E2 levels increased with an increase in the concentration of BPAF. This increase was most evident in the fish exposed to 1 mg/L BPAF. In female fish, T levels increased following exposure to 0.05 and 0.25 mg/L BPAF but decreased in the 1 mg/L treatment groups. In addition, an increase in E2 levels was observed in the 0.05 and 1 mg/L groups but a slight decrease in the 0.25 mg/L group relative to the control. Furthermore, the ratio of T/E2 in male fish decreased in a dose-dependent manner. The total FT3 levels increased in females in all exposure groups, and were significantly higher in the 1 mg/L group compared with the control.

Superoxide Dismutase Acivity (SOD) and Malondialdehyde Content (MDA)

There was no difference in SOD activity among the control and treatment groups of both genders after 28 days exposure. Similarly, exposure to BPAF had no impact on liver MDA content in males and females.

VTG Gene Expression

In males, exposure to 1 mg/L BPAF caused significant upregulation of the VTG gene in the liver. The expression of VTG tended to increase in females in all treatments, but the difference was only significant in the 0.25 mg/L treatment group.

- Type of and number with behavioural abnormalities: not reported
- Other biological observations: no
- Effect concentrations exceeding solubility of substance in test medium: not reported
- Incidents in the course of the test which might have influenced the results: not reported

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

All data are expressed as the mean6standard error of mean (SEM). SPSS statistical software (Version 13.0) and Sigma Plot 10.0 were used for statistical analysis. The significant differences between control and treated groups were determined using a one-way analysis of variance and Tukey’s multiple range test. Differences were statistically significant if P ≤0.05.

Table 1: Effect of BPAF exposure on the growth of zebrafish

Zebrafish	BPAF (mg/L)	Length (cm)	Weight (g)
Female	0	3.20 ± 0.04	0.332 ± 0.011
	0.05	3.14 ± 0.03	0.349 ± 0.012
	0.25	3.20 ± 0.03	0.350 ± 0.008
	1	3.20 ± 0.03	0.325 ± 0.009
Male	0	3.20 ± 0.02	0.287 ± 0.005
	0.05	3.18 ± 0.02	0.287 ± 0.006
	0.25	3.13 ± 0.02*	0.266 ± 0.005*
	1	3.14 ± 0.03*	0.282 ± 0.009

* P <0.05 (significant difference versus control value)

Validity criteria fulfilled:

not specified

Remarks:

Dissolved oxygen concentrations not reported.

Conclusions:

NOEC for mortality ≥ 1 mg/L (highest tested concentration). The NOEC for growth rate was determined to be 0.05 mg/L, where a significant reduction in growth (length and weight) was observed in the male fish treated at 0.25 mg/L for 28 days.

An imbalance of sexhormones was observed where testosterone levels in male fish declined in a dose-dependent manner. Concomitantly, E2 concentrations were seen to increase in a dose-dependent manner- significantly at 1 mg/L (p<0.05). As a result, VTG induction in males was seen to be significantly increased in individuals treated at 1 mg/L (p<0.01), and additionally in females treated at 0.5 mg/L (p<0.05). The NOEC for sexual hormone imbalance and/or the induction of VTG in zebrafish was 0.05 mg/L.

Executive summary:

The 28 day chronic toxicity of Bisphenol-AF (BPAF) to juvenile zebrafish (Danio rerio) was studied under semi-static conditions. 2 -month old zebrafish were exposed to nominal concentrations of 0.05, 0.25 and 1 mg/L BPAF and a solvent control (0.1 % dimethylsulfoxide). The test system was maintained at 28 ± 0.5 ºC. The 28 day NOEC value, based on mortality, was ≥ 1 mg/L, the highest tested concentration. The sublethal effects were a significant (p < 0.05) decrease in male growth, with regards to length at 0.25 and 1 mg/L and also weight at 0.25 mg/L. In addition, the effects of BPAF on sex hormone concentrations and gene expression were also evaluated in both male and female individuals. In males, testosterone concentrations in whole fish homogenates decreased in a dose dependent manner as the concentration of BPAF exposure increased. In contrast, concentrations of E2 increased in a dose dependent manner, significantly so at 1 mg/L (p < 0.05) exposure. In females, concentrations of both testosterone and E2 generally increased in a dose dependent manner, although a decrease in testosterone and E2 were observed versus the control fish at 1 and 0.25 mg/L, respectively. None of the hormone increases were statistically significant. The increase in VTG induction in males and females was also significantly increased at 1 and 0.25 mg/L versus control values, respectively, indicating potential endocrine disruption.

This toxicity study is classified as supplementary as it does not fully satisfy the OECD guideline requirements of the Fish, Juvenile Growth Test (OECD TG 215).

Results Synopsis

Test Organism: Zebrafish (Danio rerio)

Test Type: Semi-static

NOEC for mortality: ≥ 1.0 mg/L

NOEC for fish growth: 0.05 mg/L

NOEC for sex hormone deficincies/ endocrine disruption potential: 0.05 mg/L

Applicant's interpretation:

This study is considered to be similar to the Fish, Juvenile Growth Test (OECD 215, 2000), however the test did not state specific growth rates.

Two month old zebrafish (Danio rario) were maintained in a recirculating aquarium at 28 ± 0.5 °C, with a 12 hour photoperiod per day and fed twice daily with Artemia nauplii. The fish were naive to chemical pollution. The fish were exposed to a vehicle control, 0.05, 0.25 or 1 mg/L of test item. Males and females were held separately in 6 L tanks.The tanks holding water was renewed at a rate of 3 L per day. There were four replicate tanks per sex per concentration with 9 fish in each (i.e. 36 fish per concentration). Fish were acclimated for 1 week and then body weight and length were measured. Following the acclimation period, fish were exposed for 28 days. The study only reports nominal concentrations, although the chemical has been shown to stable in these systems using DMSO as a solvent, i.e. there is sufficient evidence within the literature to suggest that 1) the test item is stable in environmental matrices, 2) the chemical is sufficiently soluble at the tested level, and 3) the test item will not shift pH or have a high oxygen demand. For example Tisler et al. 2016 verified during a chronic Daphnia magna test that the test item was stable (i.e. within 90 % of nominal dosed concentrations) after 3 days in Daphnia medium (Elendt M4). The medium used here is much less complex and similar stability would be expected. Song et al. (2014) noted that the test concentrations (including DMSO) were homogenous in the test replicates during a long-term fish study, and confirmed by LC/MS. Further, DMSO appears to be an appropriate solvent in all fish testing with high rates of hatching success and survival, and minimal noting of malformations or ill health in Song et al. (2014), Tang et al. (2015) and Yang et al. (2014), which all used DMSO as a solvent. Song et al. (2014) also included a DMSO and medium control to show that no significant effect was caused by the use of the solvent. An algae study conducted by DuPont (2007) and the chronic Daphnia magna study conducted by Tisler et al. (2016) provide further evidence that water quality parameters will not be greatly affected by these concentrations of the test item.

After 28 days all fish were frozen and the weights and lengths were measured. Further, the condition of the fish was calculated using a condition factor calculation: CF = 100 x [body weight (g)/total length^3(cm)]. Histological, hormone, anti-oxidant and gene expression measures were also taken. However, under the current regulatory frameworks these endpoints are not usable for hazard assessment purposes. Although VTG gene expression was monitored, this is outwith the OECD 234 guideline (2011) measures and again gene expression measures is not currently used in hazard assessment, this is in large part as it is difficult to associate the exact whole body biologically impact of shifts in gene expression without either comprehensive data showing the minimal increase/decrease that is needed to cause an associated effect, or without direct measures at the whole organism level. The authors showed a significant but transient effect on weights of male fish at 0.25 mg/L, but as there was no obvious dose-repose and as the condition factor was not significantly impacted, this effect is not considered of biological significance and is likely within the normal range for the fish.

There was also a transient non dose-dependent effect on female fish, shown as a significant increase in condition factor at 0.05 mg/L. Again, this is not seen as biologically significant and there is no significant difference seen in weight or length at this concentration, nor is there a significant difference seen at the higher concentrations.

In males there is also a statistically significant difference in length at 0.25 and 1 mg/L, however, the purpose of the study is to determine growth rate and for this purpose weight is the critical endpoint, these minor deviations in length are considered incidental as weight was largely unaffected and nor was the condition factor. Further, considering the standard error of the controls and the exposure concentrations the difference between lengths is considered minimal and inconsequential. Although NOECs have been determined for length by the authors, this is considered a statistical misrepresentation of biological health. Further, length, weight and condition factor are highly dependent endpoints and it would be expected if there were biological significance and impact on growth that all endpoints would show significant and consistent effects.

Not all validity criteria were outlined in the test and is not possible to determine if the study mean weight of fish in the control(s) increased enough to permit the detection of the minimum variation of growth rate considered as significant, or dissolved oxygen. Although all other parameters were met: temperature was consistent and the mortality in the controls was =< 10 %.

Due to lack of biologically significant effects, lack of a clear dose response, a lack of corroboration between highly dependent endpoints and that the critical endpoint for the study as per the OECD 215 guideline is growth (measured by weight), it can be considered that the NOEC is => 1 mg/L. Therefore, no precise and definitive value can be given for the NOEC, the NOEC should be used with caution due to the concerns around the validity criteria. Furthermore as growth rate was not determined the reliability of the NOEC is uncertain and should not be used for hazard or risk assessment.

Description of key information

NOEC (post-hatch mortality) = 0.05 mg/L, Similar to OECD 204 (1984), Yang et al., 2014

NOEC (post-hatch mortality) = 1.5 mg/L, OECD 210 (2013), Song et al., 2014

NOEC (hatchability at 72 hours) => 0.125 mg/L, Similar to OECD 236, Shi et al., 2015

NOEC (fertilization rate) = 0.025 mg/L, Similar to OECD 236, Shi et al., 2015

NOEC (malformation) = 0.025 mg/L, Similar to OECD 236, Shi et al., 2015

Key value for chemical safety assessment

Fresh water fish

Dose descriptor:: NOEC
Effect concentration:: 0.125 mg/L

Additional information

Key value for CSA:

There are 3 existing studies on the long term toxicity of fish. The studies were similar to the Fish, Juvenile Growth Test (OECD 215, 2000), the Fish Sexual Devolopment test (OECD 234,1998) and the Fish Early Life-Stage Toxicity Test (OECD 210, 2013). Although these tests were similar to the guidelines, some deviations were noted.

A study by Yang et al. (2014) is considered to be similar to the Fish, Juvenile Growth Test (OECD 215, 2000), however the test did not state specific growth rates.

A study by Song et al. (2014) was conducted using similar methods to those described in the OECD guidelines 234 (2011) and 236 (2013). However, the recommended duration for the tests was not met for the OECD 234 test (21-d test compared to guideline requirements of 60 day post fertilisation (OECD 234, 2011)). Further the OECD 234 is not a standard test listed for REACH endpoint fulfilment for long-term toxicity to fish (REACH legislation Annex IX). However, the study further supports that effects on length, weight, hatching success and survival/mortality are seen above 1 mg/L as seen in the study by Yang et al. (2014).

In a study similar to an OECD 210 (2013) conducted by Shi et al. (2015), the toxicity in the early life stages of fish was assessed, further though the authors also conducted an assessment on the impact of the F1 generation.

The key endpoint from the study is an F0 NOEC of > 125 µg/L.

Conservatively, the 120 dpf NOEC from the study conducted by Shi et al. (2015) will be used in the key endpoint in the hazard assessment of this substance.

Of the studies presented, the evidence suggests the chemical may have ebdocrine disrupting properties (ED). The methods for each study have been previously described and the following outlines the methods for assessing ED and contextualises the results of these studies in terms of ED. There are four well-known MoAs for ED these are comprising of oestrogen, androgen, thyroid and steroidogen modalities (WHO, 2012 & EC, 2016). These will be the focus herein.

Endocrine Disruption:

Firstly, Yang et al. (2014) specifically studied the potential of the test item to dirupt sex hormone regulation and vitellogenin (VTG). VTG is a major precursor of egg yolk, and is synthesized in the liver of females in all oviparous vertebrates. VTG is transported through the blood to the ovary, and ultimately absorbed in the mature oocytes. VTG in adult male fish can be induced by the presence of oestrogenic compounds. Thus, the induction of VTG gene expression in males or larvae has been used as a sensitive biomarker for oestrogenic disruption. Expression of VTG is under the control of 17beta-estradiol, which can bind to specific endocrine receptors (ERs) in response to the stimulation of endocrine-disrupting chemical. Here the study focuses on the possible disturbance of the oestrogenic (VTG and estradiol; E2), androgenic (testosterone; T) and thyroid (triiodothyronine; T3). Both the oestrogenic and androgenic effects also come under steroidogenic effects. Histological observtions were also made on the gonad. Whole body homogenates of T, E2 and T3 were measured using standard biochemcil assays. VTG was measured indirectly using VTG gene expression. VTG is a standard endpoint under the OECD 234 guideline (2011) which was designed to study perturbations in fish sexual development, and potential ED. However, the study does not rely on gene expression due to the reasons previously mentioned. VTG gene expression was significantly increaed in males, although alone gene expression can not always infer biological alterations, the authors also noted acellular areas in the testis or inhibited oocyte maturation from the histological observations. Furthermore, in males E2 was signifcantly increased, again suggesting an oestrogenic effect. Statistically not significnat dose dependent decreases in T levels and T/E2 ratio. T3 was significantly increaed in female fish. Based on the evidence from the study, the test item is potentially an endocrine disruptor.

Song et al. (2014) studied the oestrogenic activity of the test item. VTG levels and oestrogenic activity were assessed. VTG was measured in the blood of exposed zebrafish and was smeasured in the sreum using comparative enzyme-linked immunoabsorbant assay (ELISA), similar to that described in the OECD guideline (OECD 234, 2011). Oestrogenic activity was direclty measured in vitro using the the MCF-7-p-Vit-tk-Luc-Neo assay which measures activity using luminesence. Breifly, This cell line was stably transfected with the luciferase reporter gene and estrogen-responsive element, and ER agonists can induce the production of luciferase. Luciferase activity is then measured on luminescence Microplate Reader after cells were exposed to test compound for 48 h, total protein content was simultaneously measured by the Bradford Blue assay to normalize luminescent units. VTG was significantly increased in a dose-dependent manner in all zebrafish exposed to the test item, the exposure concentrations were 0.5, 1.0 and 1.5 mg/L. During the MVLN assay oestrogenic activity was also increased at 10 µM of test item and showed a dose dependnet response. However, there was no impact at the whole organism level when considering endpoints which may have been due to ED, i.e. in the measured endpoint of hatching rate, although increased oedema was observed this is non-selective mode of action. Although VTG and oestrogen activity were significantly impacted it is unsure on how these shifts will impact the population or individual fish. The study provides some evidence of the test item potentially being an ED but the endpoints can not be linked to biological outcome. According to the WHO/IPCS (2002) definition, a chemical is only an ED if an adverse in vivo effect can be plausibly linked to an endocrine MoA. Further under many regualtory frameworks it is important to establish a plausible biological link between the MoA and the whole organism effect. However, there is no causal link made in this study and as such no plausible links can be directly made. The study shows that the test item is potentially an endocrine disruptor.

Shi et al. (2015) studied oestrogenic (E2) and androgenic (T) pertrubations of zebrafish expsosed to the test item. Furthermore, genes of the hypothalamus-pituitary-gonad axis were studied, which largely controls many of the hormones which may be impacted by ED chemicals (i.e. gonadotropin releasing hormone (GnRH), lutenizing hormone (LH), follicle stimulating hormone (FSH) and oestrogen and testosterone). These measures were taken after 120 -d of exposure. The hormone analysis was taken from blood samples taken from the caudal veins, the RNA was extracted from homogenates of brains, gonads and liver. The study showed that after 120-d of exposre to the test item E2 and E2/T ratio was significantly increased in both males and females in a dose-dependent manner. For males the NOEC was 5 µg/L and for females it was 25 µg/L. T significantly decreaesed in males only with a NOEC of 25 µg/L, again in a dose dependent manner. No such effect was noted in the female fish. In male fish several genes at 125 µg/L were either significantly suppressed or induced in all tissues. In the liver vtg1 significnatly increased at 25 and 1255 µg/L. Minimal gene expression changes were seen in female fish, although there was one gene which was significantly increased and decreaed (fshr and star) at 125 µg/L. Genes invovled with the regulation of GnRH (e.g. gnrh2), LH (e.g. lh-beta, lhr) and FSH (e.g. fsh-beta, fshr) were all effected, and thus the regualtion of testosterone and oestrogen may be effected. This is shown at the sub-organism level seen in the E2 and T resutls of the study. Further there is a causal link between the disruption of these genes and steriodal hormones with whole leve in vivo effects. The link is also plausable. For the F0 generation there was a decrease in ferilization rate of females, and this was significant at 125 µg/L, however this did not appear dose-dependent, possibly as higher concentrations were not tested, nevertheless, ED chemicals may not present dose-dependent relationships and therefore there is still potential that this is an affect of an ED chemical (EFSA, 2015). Further, malformation rate of offspring was also increaed ar 125 µg/L, survival was also decreaed at the highest concntration (125 µg/L). The study shows that the test item is potentially an endocrine disruptor.

The presented evidence suggests a causal link between ED specific gene and hormone disruption and biological repsonse. Based on the genes and hormones affected the link is also plausable. Considering the evidence presented, the test item is potentially an endocrine disruptor in fish.

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Registration Dossier

Registration Dossier

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Diss Factsheets

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

Long-term toxicity to fish

Administrative data

Link to relevant study record(s)

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Reference 1

Reference 2

Reference 3

Description of key information

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Additional information

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