Toxicity, Teratogenic and Estrogenic Effects of Bisphenol A and its Alternative. Replacements Bisphenol S, Bisphenol F and Bisphenol AF in Zebrafish.

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1 1 Supporting Information 2 3 Toxicity, Teratogenic and Estrogenic Effects of Bisphenol A and its Alternative Replacements Bisphenol S, Bisphenol F and Bisphenol AF in Zebrafish. 4 5 John Moreman, Okhyun Lee, Maciej Trznadel, Arthur David, Elizabeth M. Hill, Tetsuhiro Kudoh and Charles R. Tyler* 6 7 Correspondence: Charles R. Tyler, Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD 8 Tel: , C.R.Tyler@exeter.ac.uk Summary: Seven SI pages containing protocol descriptions of LC-MS used for chemical detection of bisphenolic chemicals in exposure medium and fraction taken up in 120 hour post-fertilisation (hpf) zebrafish larvae (Tables S1-S4). Two figures indicating dose response curves for mortality (Figure S1) and hatching rate (Figure S2) of 96 hpf zebrafish larvae on exposure to different bisphenolic chemicals. 14 1

2 Water Chemistry LC-MS Quantification. For each compound up to 100 ml of tank water containing 2 % of methanol and 0.1 % of acid acetic were extracted through an Oasis HLB (6 ml, 200 mg) cartridge (Waters, Manchester, UK), which was previously conditioned with 5 ml of methanol and 5 ml ultrapure water at a flow rate of 5-10 ml/min. Prior to SPE extractions, two internal standard (BPA-d8 and 2,2 -BPF) were added at a 1:1 ratio of compound/is. The cartridge was washed with 5 ml of distilled water, and then dried under vacuum, elution was subsequently performed with 5 ml of methanol. Extracts were dried, reconstituted in water/acetonitrile (7/3, v/v) and passed through 0.22 µm centrifuge filters. Recovery test of the SPE protocol performed at a low and high concentration for each compound (n=4 for each concentration) gave values ranging from 83 ± 2 to 105 ± 3% (Table S1). Ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC- MS/MS) analyses were carried out using a Waters Acquity UHPLC system coupled to a Quattro Premier triple quadrupole mass spectrometer from Micromass (Waters, Manchester, UK). Samples were separated using a reverse phase Acquity UHPLC BEH C18 column (1.7 μm particle size, 2.1 mm 100 mm, Waters, Manchester, UK) maintained at 25 C. Injection volume was 5 µl and mobile phase solvents were water (A) and acetonitrile (B) in an initial ratio (A:B) of 70:30. Separation was achieved at 25 C using a flow rate of 0.25 ml/min with the following gradient: 70:30 to 30:70 in 8 min; 30:70 to 0:100 and held for 4 min; return to initial condition for 12 min and equilibration for 6 min. Retention times, ionisation and fragmentation settings are reported in Table S2. MS/MS was performed in the Multiple Reaction Mode (MRM) using ESI in the negative mode, and one characteristic fragment of the deprotonated molecular ion [M H] was used for quantitation. Other parameters were optimised as follows: capillary voltage 3.3 kv, extractor voltage 8 V, multiplier voltage 650 V, source temperature 120 C, desolvation temperature 300 C. Argon was used as collision 2

3 gas (P collision cell: mbar), while nitrogen was used as both the nebulizing (100 L/h) and desolvation gas (600 L/h). Mass calibration of the spectrometer was performed with sodium iodide. Data were acquired using MassLynx 4.1 and the quantification was carried out by calculating the response factor of BP compounds to internal standards. Concentrations were determined using a least-square linear regression analysis of the peak area ratio versus the concentration ratio native to deuterated. Five point calibration curve (R 2 > 0.99) covered the range pg (injected on column) for all compounds, within the linear range of the instrument. Measured water concentrations for each bisphenol are shown in Table S Table S1. Recovery (%) of solid phase extraction of bisphenols from spiked ultrapure water. Compound BPA BPF BPAF BPS Spiking level (ng/spe) Recoveries Av SD Av = average; SD = standard deviation (n=4) Table S2. Retention times and MRM conditions used for UHPLC ESI-MS/MS analysis of bisphenols (negative mode) Compound RT MRM transition TQ parameter Precursor Transition Cone (V) Collision (V) BPA BPF BPAF BPS

4 Table S3. Measured water concentrations for each bisphenol. Data as shown are the mean of 3 replicate tanks, repeated 3 times (SEM in brackets) Nominal concentration (mg/l) Measured concentration (mg/l) BPA (0.0003) (0.01) (0.01) BPF (0.0003) (0.01) (0.06) BPAF (0.0001) (0.0001) (0.005) BPS (0.4) (0.3) (0.3) Internal Chemical Concentration LC-MS Quantification Analyses were performed using Surveyor MS Pump Plus HPLC pump with HTC PAL autosampler coupled to TSQ Vantage triple quadrupole mass spectrometer equipped with heated electrospray (HESI II) source (all ThermoFisher Scientific, Hemel Hempstead, UK). Chromatographic separation was achieved using reversed-phase, 3 µm particle size, C18 Hypersil GOLD column 50 mm 2.1 mm i.d. (Thermo Scientific, San Jose CA, USA). Analytes were separated using a linear gradient of (A) water and (B) methanol. The initial conditions for the gradient consisted of 10% solvent B which was increased to 100% in 4.5 4

5 min and maintained for 1 min before returning to the initial 10% B. The flow rate was 500 µl/min. The auto-sampler temperature was set at 8 C while the column was kept at ambient room temperature. HESI probe was operating in the negative mode; an ion-spray voltage of -4.0 kv was applied. The heated capillary temperature was set at 275 C and the vaporizer temperature was 60 C. Nitrogen was employed as sheath and auxiliary gas at a pressure of 30 and 5 arbitrary units, respectively. The argon CID gas was used at a pressure of 1.5 mtorr and the optimum collision energy (CE) for each transition was selected. Quantification of the target compounds was performed by monitoring two characteristic multiple reaction monitoring (MRM) transitions (Table S4) Table S4. LC-MS quantification of the target compounds performed by monitoring two characteristic multiple reaction monitoring (MRM) transitions Compound Parent ion (m/z) Product (m/z) ion Collision energy (ev) BPA BPF BPAF BPS

6 Figure S1. Mortality rates of 96 hpf zebrafish larvae exposed to bisphenol chemicals. Concentration response curves were modelled using a generalised linear model (GLM) in R. LC50 was defined as the concentration inducing 50 % mortality. Data points are mean values with error bars representing SEM. 6

7 Figure S2. Hatching success rate of 72-hpf zebrafish larvae exposed to bisphenol chemicals. Concentration response curves were modelled using a generalised linear model (GLM) in R. EC50 was defined as the concentration inducing 50 % of the maximal effect. Data points are mean values with error bars representing SEM. 7

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