Toxicokinetics of perfluorinated alkyl acids influences their toxic

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Ecotoxicology and Human Environmental Health

Toxicokinetics of perfluorinated alkyl acids influences their toxic potency in the zebrafish embryo (Danio rerio) Carolina Vogs, Gunnar Johanson, Markus Näslund, Sascha Wulff, Marcus Sjödin, Magnus Hellstrandh, Johan Lindberg, and Emma Elisabeth Wincent Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07188 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Environmental Science & Technology

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Toxicokinetics of perfluorinated alkyl acids

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influences their toxic potency in the zebrafish

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embryo (Danio rerio)

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Carolina Vogs1*, Gunnar Johanson1, Markus Näslund1,2, Sascha Wulff1,2, Marcus

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Sjödin2, Magnus Hellstrandh2, Johan Lindberg2, Emma Wincent1,2

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1

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Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden

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Swedish Toxicology Sciences Research Center (Swetox), 151 36 Södertälje, Sweden

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ACS Paragon Plus Environment

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Corresponding Author*

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Address: Institute of Environmental Medicine, Karolinska Institutet, Sweden

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Email: [email protected]

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Abstract image

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Abstract

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Perfluorinated alkyl acids (PFAA) are highly persistent and bioaccumulative and have

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been associated with several adverse health effects. The chemical structure mainly differs

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in two ways: the length of the hydrophobic alkyl chain and the type of hydrophilic end

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group. Little is known how the chemical structure affects the toxicokinetics (TK) in different

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organisms. We studied the TK of four PFAA (PFOS, PFHxS, PFOA, PFBA) with different

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chain lengths (4-8 carbons) and functional groups (sulfonic or carboxylic acid) in zebrafish

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(Danio rerio) embryo. The time courses of the external (ambient water) and internal

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concentrations were determined at three exposure concentrations from 2 up to 120 hours

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post fertilization (hpf). Three of the four PFAA showed a biphasic uptake pattern with slow

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uptake before hatching (around 48 hpf) and faster uptake thereafter. A two-compartment

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TK model adequately described the biphasic uptake pattern, suggesting that the chorion

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functions as an uptake barrier until 48 hpf. The bioconcentration factors (BCF) determined

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at 120 hpf varied widely between PFAA with averages of approximately 4000 (PFOS),

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200 (PFHxS), 50 (PFOA) and 0.8 (PFBA) L kg dry weight-1, suggesting that both the alkyl


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chain length and the functional group influence the TK. The differences in toxic potency

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were reduced by three orders of magnitude when comparing internal effect

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concentrations instead of effective external concentrations.

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Keywords

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perfluorooctanesulfonic

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perfluorobutanoic acid, toxicokinetic model, UPLC-MS/MS

acid,

perfluorohexane

sulfonate,

perfluorooctanoic

acid,

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Introduction

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Perfluorinated alkyl acid carboxylates and sulfonates (PFAA) are a group of persistent

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organofluorine chemicals with high thermal and chemical stability, which have been

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broadly used in commercial and industrial products (e.g. surfactants, fluorinated

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polymers, coatings, fire-resistant foams). PFAA are extremely persistent in the

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environment and globally distributed. PFAA contamination have been found in drinking

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water and river water1-3, soil and biota4-5 and food products6. Perfluorooctane sulfonate

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(PFOS), perfluorooctanoic acid (PFOA) and perfluorohexane sulfonic acid (PFHxS) are

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among the most frequently monitored PFAA with varying environmental concentrations

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of levels from pg L-1 to µg L-1 1, 7-10. The PFAA differ in terms of chain lengths and functional

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groups and are thus likely to vary with respect to the fate in the environment, toxicokinetics

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(TK), bioaccumulation and toxicity. However, it is challenging to predict these differences

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as many of the physicochemical properties (e.g. partitioning coefficients, acid dissociation

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constants, water solubility) are scarce or controversial and little is known about how these

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properties affect e.g. protein binding and biotransformation.


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Exposure to PFAA in the general human population occurs mainly through drinking

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water, diet and consumer products8, 11. Human whole blood concentrations measured in

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Swedish residents ranged between 1.7 - 37 µg L-1 for PFOS, 0.5 - 12.4 µg L-1 for PFOA,

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and 0.4 - 28.4 µg L-1 for PFHxS12. Much higher serum levels were detected in another

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Swedish population (PFOS 24 - 1500 µg L-1, PFOA 2.4 - 92 µg L-1, PFHxS 12 – 1660 µg

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L-1) exposed via contaminated drinking water8. Several PFAA, including PFOS, PFHxS

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and PFOA, were detected in serum samples in over 75% of the individuals in a Swedish

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cohort13. Relative levels of PFAA found in different autopsy tissues (i.e. liver, kidney, lung,

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brain, bone) indicated that the distribution in the human body varies between the PFAA,

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which is likely due to their differences in physicochemical properties14. In addition to the

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wide prevalence of PFAA in humans, elimination from the human body is very slow as

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reflected by mean half times of 3.4, 2.7 and 5.3 years for PFOS, PFOA and PFHxS,

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respectively8. Still, the TK processes, and how they differ between PFAA with different

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chain lengths and functional groups, are poorly understood.

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PFAA accumulation potential in humans may be associated to human health effects, but sparse evidence still exists15,

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To date, experimental studies have revealed that


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PFAA exposure may cause multiple adverse effects such as hepatotoxicity,

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immunotoxicity, developmental toxicity, reproductive toxicity, neurotoxicity and cancer17-

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20.

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PFAA have been shown to interfere with various signalling pathways suggesting that

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several adverse outcome pathways and cross-talks may play a role in toxicity21. Due to

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emerging health concerns, large efforts to manage potential risks associated to PFAA

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exposure are ongoing, such as the banning of longer chain-length PFAA (≥ C8) under the

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Stockholm Convention on Persistent Organic Pollutants22. As a result, longer chain-length

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PFAA have been substituted by shorter chain-length PFAA such as PFHxS and

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perfluorobutanesulfonic acid (PFBA). The shorter chain-length PFAA are assumed to be

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less bioaccumulative, eliminate faster from the human body and less harmful. However,

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experimental data confirming these assumptions are yet lacking.

Although the mechanisms underlying these adverse effects are still largely unknown,

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The zebrafish (Danio rerio) embryo (ZFE) is increasingly used as an alternative model

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to fish and other higher vertebrates for toxicity testing in risk assessment. The ZFE as

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experimental model has many advantages, e.g. a rapid ex uterus development with major

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organs functioning already at 72 h post fertilization (hpf), optical transparency in early


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developmental stages and cost-efficient usage. Thus, the ZFE model has been applied

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to study toxicity mechanisms of numerous chemicals from molecular initiating events to

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phenotypical changes. Previous studies have shown that PFAA with longer chain length

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and with a sulfonic functional group are more potent with respect to developmental toxicity

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in ZFE than those with shorter chain length and with a carboxylic functional group23-25.

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However, the differences in toxic potency could not be explained by mode of action,

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suggesting that they are due to TK differences. To date, the uptake kinetics of PFAA in

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ZFE have only been determined for PFOS26 and the TK of other PFAA as well as the

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relation with e.g. chain length and functional group remain open issues.

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The aim of this study was to describe and compare the TK profiles and bioconcentration

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factors of four PFAA in ZFE, namely the sulfonic acids PFOS (C8) and PFHxS (C6) and

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the carboxylic acids PFOA (C8) and PFBA (C4). Our hypothesis was that the different

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physicochemical properties of these PFAA have an impact on TK processes such as

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uptake, biotransformation and excretion. To this end, we measured the PFAA

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concentrations in exposure medium and in the ZFE at three exposure levels over time. In

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addition, a two-compartment TK model was applied to fit determined internal


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concentrations and estimate kinetic rates. Bioconcentration factors were calculated and

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used to estimate the internal effective concentration of the PFAA.

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Materials and Methods

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Chemicals

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All chemicals were purchased from Sigma-Aldrich (Stockholm, Sweden) unless stated

114

otherwise. CAS No. and physicochemical properties for the PFAA are listed in the

115

Supplement (SI Table S1). Chemical specific purity was ≥ 98% for PFOS, PFHxS and

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PFBA and > 96% for PFOA. Stock solutions of PFOS, PFHxS and PFOA were prepared

117

in dimethyl sulfoxide (DMSO, CAS No. 67-68-5) and PFBA was prepared in double-

118

distilled water (ddH2O, laboratory reagent ELGA Purelab flex). 3-morpholinopropane-1-

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sulfonic acid (MOPS, CAS No. 1132-61-2) and sodium hydroxide (NaOH, CAS No. 1310-

120

73-2) was added to PFBA and PFOA exposure media to prevent pH changes. HPLC

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grade acetonitrile (ACN, CAS No. 75-05-8, VWR, Stockholm, Sweden) and ddH2O were

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used for sample preparation. The isotope labelled internal standards (IS) sodium


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perfluoro-1-[1,2,3,4-13C4]octanesulfonate, perfluoro-n-[1,2,3,4-13C4]octanoic acid, sodium

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perfluoro-1-hexane[18O2]sulfonate and perfluoro-n-[1,2,3,4-13C4]butanoic acid were

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purchased from Wellington Laboratories (Guelph, Canada). E3-Medium were prepared

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using sodium chloride (NaCl, CAS No 764714-5), potassium chloride (KCl, CAS No.

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7447-40-7), calcium chloride dihydrate (CaCl2*2H20, CAS No. 10035-04-8), magnesium

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sulfate (MgSO4, Cas No. 7487-88-9) and 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic

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acid (HEPES, CAS No. 7365-45-9). For the UPLC-MS/MS analysis, mobile phases

130

consisted of methanol (MeOH, CAS: 67-56-1, Optima LC/MS GRADE, Fisher Scientific),

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acetonitrile (ACN, CAS No. 75-05-8, Optima LC/MS GRADE, Fisher Scientific) and

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ammonium acetate (NH4Ac, CAS No. 631-61-8, LC/MS LiChropur).

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Zebrafish and exposure

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ZFE of the AB strain and E3 medium (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33

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mM MgSO4, 0.2 mM HEPES) were provided by the Zebrafish Core Facility at Comparative

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Medicine, Karolinska Institutet. Three male and female adult zebrafish were paired and

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the eggs were collected directly after spawning and transferred into E3 medium. The ZFE


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batch was examined under a light microscope (Nikon SMZ1270 Stereomicroscope) to

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discard unfertilized eggs. All exposure experiments were set up according to the test

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guideline OECD TG 23627 for fish embryo toxicity testing. Briefly, embryos were exposed

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from 2 hpf up to 120 hpf. 30 embryos were added to 30 mL exposure medium (one

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embryo per mL) in 50 mL glass petri dishes. The dishes, covered with lids, were kept in

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a climate chamber at 28 ± 1 ºC under dark conditions throughout the exposures.

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Concentration-effect relationships of PFAA

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Two or three independent concentration-effect-relationship experiments were

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conducted with each PFAA to determine the concentration causing effects in 20% of the

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embryos (EC20). The EC20 values were subsequently used as the upper limit for the

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highest exposure concentrations in the TK experiments. Effects on morphology were

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assessed at 120 hpf using a Nikon SMZ25 microscope and included lethal (coagulation,

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lack of heartbeat, non-detachment of tail, non-hatching and lack of somite formation) as

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well as sublethal (non-inflated swim bladder, pericardial and yolk sac edemas, scoliosis)

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apical endpoints. A ZFE with any of these morphological changes was considered to be


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affected. Starting at 2 hpf, fertilized ZFE were exposed to a wide range of concentrations

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of respective PFAA (Table 1). To avoid effects from altered pH, the exposure media with

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PFOA and PFBA were buffered with 10 mM MOPS and adjusted to pH of 7.4 with 5 M

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NaOH23. Concentration-effect relationships were obtained by fitting the sigmoidal Hill

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model (eq. 1) to respective PFAA data set using the GraphPad software (version 5.02,

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La Jolla, USA) 𝐶𝑛

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𝐸 = 𝐸𝑚𝑖𝑛 +(𝐸𝑚𝑎𝑥 ― 𝐸𝑚𝑖𝑛) × (𝐸𝐶𝑛

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E represents the percentage of ZFE affected at concentration C, Emin and Emax the

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minimum and maximum percentage affected, n the slope of the curve and EC50 the

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concentration causing an effect in 50% of the ZFE. The parameters resulting in the best

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fit to the experimental data were subsequently used to calculate the exposure

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concentration causing an effect in 20% of the ZFE (EC20) (Table 1).

50

+ 𝐶𝑛)

Eq. 1

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Toxicokinetic experiment

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TK experiments were performed in order to determine “internal concentration” and

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compare “internal effective concentration” of PFAA which are apparent values based on


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the total chemical mass measured in the whole embryo, and includes PFAA bound e.g.

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to the surfaces of the skin and chorion in addition to PFAA in ZFE tissue. Each TK

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experiment consisted of three different exposure scenarios performed in parallel as

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outlined in Figure 1. First, 30 ZFE were incubated in 30 mL E3 medium without addition

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of PFAA as a control for vitality of the ZFE batch, referred to as “biological control”.

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Second, triplicates of 30 mL exposure medium for three different concentrations of

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respective PFAA were prepared in E3 medium and incubated in glass petri dishes without

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adding ZFE, referred to as “ZFE-free medium”. The ZFE-free medium was used to check

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for unspecific loss due to e.g. adsorption to glass surfaces. Third, fertilized ZFE were

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exposed to three concentrations of each PFAA in E3 medium, hereafter called “ZFE

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medium”. All ZFE exposures were performed with 30 embryos and 30 mL medium. The

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high concentration (C1) was selected based on the EC20 values and the mid (C2) and low

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(C3) concentrations were 10 and 50 times dilutions of C1, respectively (Table 2). ZFE

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and medium were sampled at 0 (ZFE-free medium only), 3, 6, 9, 24, 31, 48, 72, 96 and

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120 hpf. Regarding medium samples, 300 µL aliquots were taken in three replicates from

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each petri dish and transferred to amber glass vials (2 mL, Agilent, Stockholm, Sweden).


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Regarding ZFE, five replicates each consisting of five embryos were collected at each of

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the above time points. The ZFE were transferred to microtubes (1.5 mL, Eppendorf Safe-

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Lock tubes, Sigma-Aldrich, Stockholm, Sweden) and excess medium was removed by

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pipetting. The ZFE were immediately washed twice with 300 µL ddH2O for a short time

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period of maximum 5 min in total to minimize back diffusion of PFAA. Subsequently, all

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samples were stored at -80ºC until sample preparation and chemical analysis. Two

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independent TK studies were conducted using ZFE from different fish batches.

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Sample preparation

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All samples were thawed at room temperature before sample preparation. Chemical-

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specific internal standards (IS) were added to all samples prior to extraction to correct for

197

possible matrix effects and/or variations in extraction efficiencies (Figure 1). Defrosted

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medium samples (300 µL) were treated by adding 700 µL of 100 nM IS dissolved in ACN

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(100%). Prior to ZFE sample preparation, 300 µL of IS solution (100 nM IS in ACN

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dissolved in ddH20 (v/v 70:30%)) was added. Thereafter, the ZFE were homogenized at

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room temperature by sonication (up to 4 times for 5 seconds to prevent evaporation, MSE


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Soniprep 150, Measuring and Scientific Equipment, London, UK), incubated at room

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temperature for 1 h and centrifuged for 10 min at 14000 rpm. The supernatants were

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transferred to amber glass vials and stored at -80 ºC prior to chemical analysis. Extraction

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recovery and matrix effect analysis are described in the Supplement.

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Chemical analysis

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All chemical analyses were performed using UPLC-MS/MS. Injection volumes were 10

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µL, 1 µL, 2 µL and 1 µL for PFOS, PFHxS, PFOA and PFBA, respectively. The PFAA

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were separated on an Acquity™ (Waters, Milford, MA, USA) on a 50 x 2.1 mm BEH C18

211

column (Waters, Sollentuna, Sweden) with gradient elution (solvent A: 2% MeOH, 1 mM

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NH4Ac in H2O; solvent B: 1 mM NH4Ac in ACN) using two separate gradient methods (SI

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Table S2). The mass spectrometer was a Xevo™ TQD (Waters), which operated in

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multiple reaction monitoring modes with negative ion mode electrospray ionization [M-H].

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A detailed description of the compound specific settings can be found in Supplement

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(Table S2). Quantification was achieved by external PFAA calibrators (n ≥ 5) prepared in

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ACN:ddH20 (v/v 70:30%) with ranges of 6-800 nM for PFOS, 8-25000 nM for PFHxS, 11–


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2800 nM for PFOA and 16–2050 nM for PFBA. Limits of quantification, defined as ten

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times the signal-to-noise ratios, were 6 nM, 25 nM, 6 nM and 18 nM for PFOS, PFHxS,

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PFOA and PFBA, respectively. The method error of concentrations expressed as the

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coefficient of variance (CoV) were 13% for PFOS, 14% for PFHxS, 13% for PFOA and

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32% for PFBA in average (SI Table S3). The large CoV for PFBA resulted from large

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variation of measured ZFE concentrations per time point and concentration.

224 225

Data analysis

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The results from analyses of medium were combined from two independent

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experiments, each consisting of three replicates per concentration and time point. The

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PFAA amount per embryo was determined by dividing the total amount of respective

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PFAA in ZFE extract with the number of embryos used per extract. The internal amount

230

in ZFE was thereafter based on volume in µmol per liter (µM) ZFE, dry weight in µmol per

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kg dry ZFE and total protein content in pmol per µg ZFE protein at respective ages (SI

232

Figure S1) to calculate internal concentrations. ZFE volume and dry weight at different

233

ages were taken from Brox et al.28, and total protein content was determined using the


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Coomassie (Bradford) Protein Assay Kit according to the manufacturer’s protocol

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(Thermos Scientific, Rockford, USA). Data from ZFE samples are presented as means of

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ten replicates for each concentration and sampling time point, originating from two

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independent experiments. Outliers were excluded using the Grubb’s test.

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Toxicokinetic characterization

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Net uptake rates were calculated as the derivative of internal amount [pmol h-1]. To

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quantify the total accumulated amount of PFAA over the exposure time. The area under

242

the curve (AUC [µM h]) was determined using the trapezoidal method and a Monte Carlo

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simulation was applied to calculate standard errors (SE) (n = 500) (Berkley Madonna,

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Version 8.3.18, California, USA). Bioconcentration factors (BCF) were determined as 𝐶𝑖𝑛𝑡

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𝐵𝐶𝐹 =

𝐶𝑤 ,

Eq. 2

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where Cint and Cw are the average concentrations in ZFE and medium, respectively, at

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120 hpf. BCF values were calculated based on both embryo volume28 (BCFvol, unitless)


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and dry weight28 (BCFdry, L kg-1). Internal effect concentrations (IEC50 [µM]) were obtained

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by multiplying the average BCFvol with the EC50 value:

250

𝐼𝐸𝐶50 = 𝐵𝐶𝐹𝑣𝑜𝑙 × 𝐸𝐶50

Eq. 3

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Kinetic rates were estimated by fitting one- and two-compartment models to the ZFE

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concentrations using Berkley Madonna. The one-compartment model included uptake

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(kWE [h-1]) and elimination (kEW [h-1]) kinetic rates for exchange processes between ZFE

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medium (Cw) and ZFE (Cint) neglecting the chorion as transport barrier.

255

𝑑𝐶𝑖𝑛𝑡

256

A two-compartment model was developed in order to implement uptake (kWC [h-1]) and

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elimination (kCW [h-1]) kinetic rates for exchange processes between ZFE medium and

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chorion (Cc) and uptake (kCE [h-1]) and elimination (kEC [h-1]) kinetic rates for exchange

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processes between ZFE chorion and ZFE until hatching at 48 hpf. Therefore, a step

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function was implemented to mimic ZFE with a chorion before hatching and ZFE without

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chorion fully exposed to PFAA after hatching.

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𝑑𝑡

𝑑𝐶𝑐 𝑑𝑡

Eq. 4

= 𝑘𝑊𝐸 × 𝐶𝑊 ― 𝑘𝐸𝑊 × 𝐶𝑖𝑛𝑡

=

𝑘𝑊𝐶 × 𝐶𝑤 ― 𝑘𝐶𝑊 × 𝐶𝑐 + 𝑘𝐸𝐶 × 𝐶𝑖𝑛𝑡 ― 𝑘𝐶𝐸 × 𝐶𝑐 𝑡 ≤ 48ℎ 0 𝑡 > 48ℎ

Eq. 5


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263 𝑘𝐶𝐸 × 𝐶𝑐 ― 𝑘𝐸𝐶 × 𝐶𝑖𝑛𝑡 =𝑘 ×𝐶 ―𝑘 ×𝐶

𝑡 ≤ 48ℎ 𝑡 > 48ℎ

264

𝑑𝐶𝑖𝑛𝑡

265

To compare the fits of the one-compartment model and the two-compartment model to

266

the observed internal concentrations, we calculated the Akaike Information Criteria (AIC

267

[-])29.

𝑑𝑡

𝑊𝐸

𝑤

𝐸𝑊

𝑖𝑛𝑡

∑(𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 ― 𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑)2

Eq. 6

268

𝐴𝐼𝐶 = 𝑁 × 𝐿𝑛(

269

with N representing sample size and Npar representing number of parameters.

𝑁

+2 × 𝑁𝑝𝑎𝑟)

Eq. 7

270 271

Results and Discussion

272 273

PFAA concentration-effect relationships

274

We first determined concentration-effect relationships for the four PFAA as too high

275

concentrations may influence the TK processes due to embryonic toxicity. ZFE exposed

276

to a wide range of concentrations (Table 1) were followed up to 120 hpf with regards to

277

aberrant morphology. Aberrant effects were observed mainly at 96 and 120 hpf with non-


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inflated swim bladder and scoliosis as the most prominent sublethal endpoints (SI Figure

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S2). The toxic potency of the PFAA, determined at 120 hpf, differed by 3000-fold with

280

PFOS being most toxic followed by PFHxS and PFOA (Figure 2). PFBA showed no

281

significant toxicity even at the highest concentration (70 mM). Moreover, all embryos

282

exposed to the highest concentrations of PFOS or PFHxS were affected, whereas 100%

283

affected ZFE was not reached at any tested concentration of PFOA or PFBA. EC50 values

284

were estimated to be 3.8, 84.5 and 509 µM for PFOS, PFHxS and PFOA, respectively.

285

For PFBA, the EC50 value could not be determined due to lack of toxicity. These data

286

indicate that both the length of the fluorinated carbon chain (8, 6, or 4 carbons) and the

287

functional group (sulfonic or carboxylic acid) of PFAA influence the toxicity. While there

288

are no reports on the toxicity of PFHxS in ZFE, the EC50 values for PFOS and PFOA

289

obtained in our study are similar to those previously reported (Table 1). Furthermore, our

290

data confirm the previous notation that PFAA with a sulfonic acid group are more toxic to

291

ZFE than those with a carboxylic acid group23-25. These authors could however not explain

292

the mechanisms underlying the previously observed differences in PFAA toxicity, but

293

suggested this might be due to TK variability. Hagenaars et al.30 reported that effects


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caused by PFOS exposure depend on the duration of exposure suggesting that a certain

295

accumulated concentration over time in ZFE is essential to cause the observed adverse

296

effects.

297 298

Time-course of PFAA concentration in exposure medium

299

To minimize confounding effects of toxicity on TK processes, the highest exposure

300

concentrations were set equal to or below the EC20 values (Table 2). Accordingly, the

301

highest concentrations differed by four orders of magnitude between the four PFAA. In

302

total, three exposure concentrations were selected for respective PFAA, encompassing

303

low to high levels compared to human exposure levels6-13.

304

To check for unspecific losses of PFAA due to e.g. adsorption to glass surface, we first

305

determined the concentration of respective PFAA over time in ZFE-free medium. All

306

concentrations of PFHxS, PFOA and PFBA remained constant over the 120 h exposure

307

while PFOS concentrations decreased within the first 3 h of exposure (SI Figure S3).

308

Measurements of ZFE-free medium showed highest variation at the lowest


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concentrations of PFOS and PFHxS and at the highest concentrations of PFOA and

310

PFBA (SI Table S3).

311

We next determined the time course of PFAA concentration in the ZFE medium (Figure

312

3A). The PFFA concentrations in the medium remained stable throughout the 120 h

313

exposure, with minor exceptions. ZFE medium concentration time courses did not differ

314

from those observed with the ZFE-free medium. (SI Figure S3). The lack of a clear

315

decrease indicates that the amount of PFAA absorbed by the ZFE is negligible relative to

316

the total amount of PFAA in the exposure medium. In contrast to our finding, Kühnert et

317

al.31 described chemical depletion of polycyclic aromatic hydrocarbons (PAH) in ZFE

318

medium over time. While the different exposure regimen used in the PAH study may

319

underlie some of the observed differences, the most likely explanation is the differences

320

in physicochemical properties, e.g. a much higher lipophilicity (log Kow 3.3 – 5.8)

321

compared to the PFAA in our study (measured log Kow 0.5 – 2.4, SI Table S1).

322 323

Time-course of PFAA concentration in ZFE


22

Page 23 of 53


324

Absorption in the ZFE was detected for all PFAA and concentrations (Figure 3B and

325

3C). The time courses of accumulated amount did not differ in shape when normalized to

326

volume, dry weight or total protein content at different ZFE ages, respectively (SI Figure

327

S4). PFOS, PFHxS and PFOA showed biphasic uptake patterns with an initial slow

328

increase of internal concentrations up to 31 hpf, followed by faster increase. In contrast,

329

PFBA reached nearly maximum internal concentrations already within approximately 24

330

hpf and showed a very slow increase afterwards. The average net uptake rates (pmol h-

331

1)

332

PFOA, which reflects the biphasic uptake pattern (Figure 4). The uptake rate of PFBA

333

showed high fluctuations at the different time points. To our knowledge, a biphasic uptake

334

pattern of chemicals in the ZFE has not previously been reported, the one exception being

335

PFOS26. Furthermore, there are no published reports comparing the uptake of PFAA with

336

different chain lengths and functional groups in ZFE. In the few TK studies on ZFE

337

published to date, time-profiles of internal concentrations have been described as either

338

a very fast uptake (similar to PFBA in our study) for polar compounds or a monophasic

339

uptake until steady-state for nonpolar chemicals28,

were 0.4-4.8 times higher from 31 hpf for all three concentrations of PFOS, PFHxS and

31, 32.

In these studies, the uptake


23


Page 24 of 53

340

kinetics were proposed to depend mainly on diffusion processes and lipophilicity

341

(disregarding biotransformation). The continuous increase in internal concentration over

342

time of PFOS, PFHxS and PFOA suggests that biotransformation is lacking or of minor

343

importance. This is in line with the literature showing no evidence that PFAA are

344

biotransformed in fish or other species.

345

The initial slow uptake rate of PFOS, PFHxS and PFOA suggests that the ZFE chorion

346

functions as a transport barrier (Figure 4). The chorion with the perivitelline space, which

347

surrounds the embryo, serves as barrier for macromolecules but is permeable enough to

348

allow supply of oxygen and nutritients33. PFAA are negatively charged organic chemicals

349

at pH 7 characterized by very low pKA values (SI Table S1), which can potentially lead to

350

repulsion from the chorion. Furthermore, PFAA have a high affinity to proteins and

351

transporters that could enable chorion binding to e.g. N-linked glycoproteins34. Brox et

352

al.35 found 5 to 30 ng higher concentrations of polar compounds in ZFE with chorion

353

compared to dechorinated ZFE, concluding that the internal concentration in the ZFE with

354

chorion was overestimated until the ZFE were hatched.


24

Page 25 of 53


355

Lowering exposure concentrations of PFAA resulted in lower internal concentrations.

356

As demonstrated by the area under the concentration-time curve (AUC, Table 2), an

357

approximately ten times concentration dilution from high to mid exposure concentration

358

led to 6, 9.5, 2.4 and 5.8 times decrease in AUC for PFOS, PFHxS, PFOA and PFBA,

359

respectively. In contrary, an approximately two times dilution from mid to low exposure

360

concentration resulted in a proportional decrease of AUC and similar net uptake rates

361

(Figure 4). This finding suggest concentration-dependent TK processes of PFAA in ZFE.

362

Our findings agree with Chen et al.36 who reported that the BCF values of long-chain

363

PFAA (PFDoA, PFTrDA, PFTeDA) in adult zebrafish were significantly higher at low than

364

at high concentrations, likely as a result of concentration-dependent TK processes.

365

Various studies36-38 emphasized that concentration-dependent TK process is relevant for

366

PFAA due to saturable protein-mediated uptake mechanisms in adult fish in addition to

367

passive diffusion. PFAA specifically bind to numerous proteins (e.g. apolipoprotein or

368

albumin in plasma, fatty acid binding proteins in liver and organic anion transporters in

369

kidney) in fish and other species.


25


Page 26 of 53

370

PFAA internal concentrations increased over time without reaching apparent steady-

371

state for any PFAA or concentration as indicated by positive net uptake rates at 120 hpf

372

(Figure 4). However, the net uptake rate decreased between 96 hpf and 120 hpf, the

373

decrease being more pronounced at high exposure concentrations. Maximum internal

374

concentrations were similar between PFAA with the sulfonic end group (PFOS and

375

PFHxS) and between PFAA with the carboxylic end group (PFOA and PFBA) even when

376

the exposure concentrations differed by ten times. This result corresponded with

377

approximately ten times lower net uptake rates of PFOS and PFHxS compared with those

378

of PFOA and PFBA suggesting different bioconcentration potentials between PFAA with

379

different functional groups.

380 381

Bioconcentration factors (BCF)

382

Bioconcentration factors varied four orders of magnitude between the four PFAA and

383

were higher for PFAA with sulfonic acid end group than those for PFAA with carboxylic

384

acid end group (Table 2). In addition, the high exposure concentration yielded lower BCF

385

values for all four PFAA compared to mid and low exposure concentrations. Thus, the


26

Page 27 of 53


386

BCFvol were similar at the mid and low concentrations, but 1.5, 1.5, 4.9 and 1.7 times

387

lower for PFOS, PFHxS, PFOA and PFBA, respectively, at the high concentrations.

388

These results again suggest saturable protein binding at the high concentrations.

389

Using the ZFE, we could confirm that BCF values increased with carbon chain length

390

as previously reported for adult fish39-43. In addition, our BCFdry values were in the range of

391

reported BCF values stated for different adult fish species (BCFdry 3.1 – 3200 L kg -1, Table 2).

392

The study by Hung et al.26 on PFOS is of particular interest as they also used ZFE.

393

However, we observed two orders of magnitude higher BCFvol for PFOS compared to

394

Hung et al.26. The discrepancy may be due to different exposure conditions, e.g. five

395

embryos per mL medium by Hung et al.26 versus one embryo per mL medium in our study.

396

Since steady-state concentrations in the ZFE were not reached at 120 hpf for all PFAA,

397

the BCFvol likely underestimates the “true” steady-state BCF.

398

For adult fish, it has been shown that PFAA dominantly accumulate in the liver, blood,

399

kidney and intestine, while approximately ten times lower concentrations are found in the

400

brain, ovary, gall bladder and muscle42-45. At present, it is not possible to determine the

401

tissue distribution of chemicals within the ZFE. Uhlag et al.46 demonstrated that exposure


27


Page 28 of 53

402

of PFAA with longer carbon chain length and with sulfonic group caused larger changes

403

in locomotor responses suggesting PFAA structure-dependent accumulation in ZFE brain

404

tissue. Overcoming the knowledge gap on PFAA tissue distribution in ZFE would increase

405

the understanding of organ-specific toxicity (i.e. developmental neurotoxicity, chemical-

406

induced hepatotoxicity) and enable comparison of target specific concentrations in other

407

species.

408

Toxicokinetic Modeling

409

We fitted a one- and a two-compartment TK model to the observed internal

410

concentrations in ZFE in order to estimate uptake and elimination kinetic rates (SI Figure

411

S5, Table S4). The one-compartment model did not adequately describe the curvature of

412

the concentration-time data but rather predicted a constant increase in internal

413

concentration without reaching steady-state. Meanwhile, the two-compartment model

414

with inclusion of chorion as a transport barrier addressed the curvature and gave a better

415

fit to the experimental values. Time trends of the fitted data from each independent TK

416

experiment were consistent and differences are mainly caused by inter-variability (SI

417

Figure S6). The Akaike Information Criteria (AIC) gave further support for the two-


28

Page 29 of 53


418

compartment model (lower AIC values), especially for the two higher PFAA exposure

419

concentrations (SI Table S4). Modelling the time courses for PFBA (all three

420

concentrations) and for lowest concentration (C3, all PFAA) generally gave less god fit

421

and less clear preference for the two-compartment model, possibly due to larger method

422

errors. Another shortcoming may be that non-linear processes such as growth and

423

concentration-dependent uptake were not included in the model.

424

The two-compartment model described a slow uptake of PFOS, PFHxS and PFOA

425

before hatching at 48 hpf and a faster uptake after hatching, in agreement with our

426

observations of internal concentrations (Figure 3C). The two-compartment model was

427

also able to match the fast increase of internal PFBA concentrations before hatching. The

428

uptake rate (kWE) differed by several orders of magnitude between PFAA (PFOS > PFHxS

429

> PFOA > PFBA), while the elimination rate (kEW) only differed one order of magnitude

430

except for PFHxS (SI Table S4). For the mid and low PFHxS exposure concentrations,

431

we estimated much lower elimination rates as a consequence of the continued stable net

432

uptake rate between 96 hpf and 120 hpf (Figure 4). Although these rate constants should

433

be interpreted with caution, it seems that the uptake rate depends more on the carbon


29


Page 30 of 53

434

chain length and functional group than does the elimination rate, in agreement with the

435

findings by Chen et al.36. We did however not find any general pattern for the PFAA uptake

436

and elimination rates between water and chorion and between chorion and embryo.

437 438

Implication for PFAA toxicity

439

Recalculation of external effect concentrations (EC) to internal effect concentrations

440

causing 20% (IEC20) in ZFE reduced the variability in toxic potency between PFAA from

441

3000-fold to 3-fold (Figure 5, Table 2). The incorporation of BCFvol thus resulted in a shift

442

in toxicity ranking from PFOS > PFHxS > PFOA > PFBA using EC20 values to PFBA >

443

PFOS ~ PFHxS ~ PFOA using IEC20 values. Altogether, this indicates that the functional

444

group as well as the carbon chain length influence TK and therefore the toxicity. In

445

agreement, Gomis et al.47 also showed that TK differences explained most of the PFAA

446

variability between concentration-effect curves of liver enlargement in male rats.

447

Moreover, the intrinsic toxic potential differed only 3-fold between PFAA indicating that

448

the toxic mechanism of action is the same for the PFAA tested. A toxic ratio analysis using

449

our calculated IEC50 values revealed that PFOS, PFHxS and PFOA are 100 times higher


30

Page 31 of 53


450

in toxicity than baseline toxicants48 (SI Figure S7). This result is in agreement with

451

literature showing PFAA specifically bind to receptors such as peroxisome proliferator-

452

activated receptors49 or estrogen receptors50 suggesting a specific mechanism of action.

453

Altogether, this indicates that toxicity differences in ZFE can dominantly be explained by

454

differences in TK rather than differences in toxic mechanisms of action.

455

Our findings and those by Gomis et al.47 indicate that the intrinsic toxic potency of short-

456

chain and long-chain PFAA may be similar. Long-chain PFAA are currently substituted

457

by short-chain PFAA (e.g. PFHxS, PFBA and Gen-X) based on the view that short-chain

458

PFAA are less toxic. As PFAA are environmentally persistent, short-chain and long-chain

459

PFAA will coexist in the environment. Meanwhile, the short-chain PFAA are more

460

bioavailable and the long-chain PFAA are more bioaccumulative. This leads to the issue

461

of combined exposure to several PFAA. Wen et al.45 reported that the bioconcentration

462

potential of short-chain PFAA was reduced in adult zebrafish when exposed in the

463

presence of long-chain PFAA, likely due to competition for transporters and binding sites

464

of proteins. However, combined PFAA exposure was shown to be more toxic to ZFE

465

compared to single PFAA exposures51. Furthermore, combined PFAA exposure resulted


31


Page 32 of 53

466

in complex interactive effects, including concentration-dependent additive, synergistic

467

and antagonistic mixture effects52, suggesting that the competition for transporters and

468

binding sites of proteins seen in adult zebrafish also occurs in ZFE. The ZFE model may

469

thus be useful to compare and better understand the influence of mixed PFAA exposure

470

on TK processes and toxicity. Such understanding is essential in the study design of

471

mixed exposure experiments.

472

Acknowledgement

473

The study was supported by a grant from the Swedish Research Council Formas (2014-

474

475

1454). We are thankful to the zebrafish core facility, Karolinska Institutet.

Supporting Information

476

Descriptions of extraction recovery and matrix effects

477

Figure S1 – S7: ZFE growth data; phenotypes of PFAA exposed ZFE; ZFE-free medium

478

and ZFE-medium concentrations; internal concentrations based on different biological


32

Page 33 of 53


479

parameters; fits of one- and two compartment model; fits of two-compartment model for

480

each individual TK experiment; toxic ratio analysis

481

Table S1 – S4: physicochemical PFAA characterization; PFAA specific settings for

482

UPLC-MS/MS; coefficient of variance; kinetic parameter estimations of one- and two-

483

compartment model

484 485 486

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Williams, D. E., Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with

634

human and rainbow trout estrogen receptors in vitro. Toxicol Sci 2010, 120, (1), 42-58.


43


Page 44 of 53

635

51. Rainieri, S.; Conlledo, N.; Langerholc, T.; Madorran, E.; Sala, M.; Barranco, A.,

636

Toxic effects of perfluorinated compounds at human cellular level and on a model

637

vertebrate. Food Chem Toxicol. 2017, 104, 14-25.

638

52. Ding, G.; Zhang, J.; Chen, Y.; Wang, L.; Wang, M.; Xiong, D.; Sun, Y., Combined

639

effects of PFOS and PFOA on zebrafish (Danio rerio) embryos. Arch Environ Contam

640

Toxicol 2013, 64, (4), 668-675.


44

Page 45 of 53

641


Figures

and

Tables

642 643

Figure 1: Workflow of sample collection and sample preparation used in the TK

644

experiment (5x5 illustrates in the figure that five replicates of five ZFE were sampled per

645

time point and concentration).


45


Page 46 of 53

646

PFOS PFHxS PFOA PFBA

Affected ZFE [%]

100 80 60 40 20 0 0.1 647

1

10 100 1000 Concentration [µM]

10000

648

Figure 2: Concentration-effect relationships for the PFAA show up to four orders of

649

magnitude differences in toxicity. Points represent the percent embryos that either were

650

dead or had one or several visible adverse effect such as non-inflated swim bladder,

651

pericardial or yolk sac edemas, and scoliosis. The curves indicate the best fit to the Hill

652

equation.


46

Page 47 of 53


B concentration in embryo (log scale)

653

0.1

0

24

48

72

96

10 1 0.1

120

0

24

48

72

96

0

24

48

72

96

1000

10 1 0

24

48

72

96

24

48

72

96

10000

10

0

24

48

72

96

0

24

48 72 96 Time [h]

120

0

24

48

72

96

120 C1=7.8 µM C2=0.8 µM C3=0.6 µM

100 50

0

24

48

72

96

120

200

0

24

48

72

96

120

300

100 10 1

C1 = 340 µM C2 = 41 µM C3 = 21 µM

400

0

120

1000

1000

100

100

1

120

50

600

Conc in ZFE [µM]

Conc in ZFE [µM] 0

100

0

120

1000

100

10

100

0.1

120

C1=0.76 µM C2=0.08 µM C3=0.04 µM

150

Conc in ZFE [µM]

Conc in ZFE [µM]

1

150

0

120

1000

10

0.1

100

Conc in ZFE [µM]

0.01

1000

C concentration in embryo (linear scale) Conc. in ZFE [µM]

Conc. in ZFE [µM]

1

Conc in ZFE [µM]

Concentration [µM] Concentration [µM] Concentration [µM] Concentration [µM]

PFBA

PFOA

PFHxS

PFOS

A concentration in ZFE medium

0

24

48 72 96 Time [h]

120

C1=4800 µM C2=550 µM C3=240 µM

200 100 0

0

24

48 72 96 Time [h]

120

654

Figure 3: Time courses of PFAA concentrations in A) ZFE medium (n=6) and in ZFE

655

depicted on B) logarithmic scale and C) linear scale (n=5-10). The exposure

656

concentrations in the legends (C1 - C3) are given as measured initial concentrations. The

657

concentrations in the ZFE [µM (=µmol L-1 ZFE)] were obtained by dividing the measured

658

amount by published data on ZFE volume at different ages (according to Brox et al.28).


47


Page 48 of 53

The lines represent simulated internal concentrations using a two-compartment model.

660

All values are stated as mean ± SE.

120

Net uptake rate [pmol h -1/Embryo]

PFOS

120




659

0.8 0.6 0.4 0.2 0.0 -0.2

0

24

48

72

96

PFOA

4

2

0 0

24

48 72 Time [h]

96

PFHxS

0.8

C1 C2 C3

0.6 0.4 0.2 0.0 -0.2

0

24

48

72

96

120

24

48 72 Time [h]

96

120

PFBA

8 6 4 2 0 -2 -4 0

661 662

Figure 4: PFAA net uptake rates per individual embryo determined as derivative of

663

internal amount at three exposure concentrations (C1 - C3).

664 665 666


48

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PFOS PFHxS PFOA PFBA

Affected ZFE [%]

100 80 60 40 20 0 667

1

10 100 1000 10000 100000 Internal concentration [µM]

668

Figure 5: Concentration-effect relationship based on internal concentrations for the PFAA

669

show up to three fold toxicity differences. Internal effect concentrations were gained by

670

multiplying BCFvol with effective exposure concentrations.The curves indicate the best fit

671

to the Hill equation based on the calculated internal effect concentrations.

672


49


Page 50 of 53

673 674

Table 1: External concentration range tested for the studied PFAA and the estimated

675

effective external concentration at which 20% (EC20) and 50% (EC50) of the ZFE were

676

affected at 120 hpf. The EC20 and EC50 values are given as mean ± SE.

Present Study

Literature values

External concentration range [µM] PFOS

0.30 - 57

EC20

EC50

EC50

[µM]

[µM]

[µM]

2.3 ± 0.1

3.8 ± 0.1

2.0 a 2.8 b

PFHxS

0.4 - 330

59.8 ± 1.9

84.5 ± 1.4

-

PFOA

8.8 - 1400

355.6 ± 5.4

509 ± 5.5

270 a 840b

PFBA

3500 - 12000

~ 7000

-

> 14000 a 10200 b

677

a

Hagenaars et al.23 (effect values at 120 hpf), b Uhlaq et al.25 (effect values at 144 hpf)


50

Page 51 of 53


678

Table 2: Initial exposure concentrations (Cw), areas under the concentration-time curve

679

(AUC), bioconcentration factors (BCFvol, BCFdry) and internal effect concentrations (IEC)

680

of the four PFAA studied at three exposure concentrations (C1 - C3). The IEC20 and IEC50

681

values were calculated as BCFvol x EC20 and BCFvol x EC50, respectively. The

682

experimental values from the present study are represented as mean ± SE.

Present study

PFOS

PFHxS

PFOA

PFBA

Literature values*

Present study

Cw

AUC

BCFvol

BCFdry

BCFdry

IEC20

IEC50

[µM]

[µM h]

[-]

[L kg-1]

[L kg-1]

[µM]

[µM]

C1

0.76 ± 0.01

8124± 18

240 ± 4

2600 ± 63

877 ± 189

1415 ± 301

C2

0.08 ± 0.01

1344 ± 13

370 ± 82

3900 ± 866

15 a 3200b 1100c 720d 2700f

C3

0.04 ± 0.002 7.8 ± 0.04

638 ± 18

510 ± 92

5400 ± 968

8483 ± 23

15 ± 0.7

160 ± 8

550b 9.6c 380f

1139± 134

1608 ± 184

170b 4.0c 3.1d 20 – 30e 11f

1805 ± 645

2584 ± 932

13b

~540

-

C1 C2

0.8 ± 0.01

893 ± 0

22 ± 0.5

230 ± 5

C3

0.6 ± 0.1 340 ± 24

762 ± 6

21 ± 3.8

220 ± 40

37924 ± 140

1.4 ± 0.1

15 ± 1

C1 C2

41 ± 2

15652 ± 55

6.9 ± 0.6

73 ± 8

C3

21 ± 1

6.8 ± 0.4

72 ± 4

C1

4800 ± 1050

10436 ± 41 19383 ± 151

0.04 ± 0.01

0.5 ± 0.1


51


C2

550 ± 31

3344 ± 31

0.07 ± 0.01

0.8 ± 0.1

C3

240 ± 14

1710 ± 18

0.10 ± 0.01

1.2 ± 0.1

Page 52 of 53

683 684 685

et al.26 – zebrafish embryo, b Chen et al.36 – adult zebrafish, c Martin et al.39 – carcass of the rainbow trout (85 % - 96 % of total mass of chemical), d Inoue et al.40 – Cyprinus carpio L., e Uhlaq et al.42 – adult zebrafish, f Naile et al.41 – fish

686

* Whole-body BCFdry values for adult fish varied by one to two orders of magnitude for

687

the same PFAA, probably due to differences in exposure systems, exposure

688

concentrations, species and gender.

a Hung


52

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For TOC art only


Toxicokinetics of perfluorinated alkyl acids influences their toxic

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