Combined ion-trapping and mass balance models to describe the pH

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Ecotoxicology and Human Environmental Health

Combined ion-trapping and mass balance models to describe the pH-dependent uptake and toxicity of acidic and basic pharmaceuticals in zebrafish embryos (Danio rerio) Lisa Bittner, Nils Klüver, Luise Henneberger, Marie Muehlenbrink, Christiane Zarfl, and Beate I. Escher Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02563 • Publication Date (Web): 09 Jun 2019 Downloaded from http://pubs.acs.org on June 9, 2019

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Environmental Science & Technology

1

2

COMBINED ION-TRAPPING AND MASS BALANCE MODELS TO DESCRIBE

3

THE PH-DEPENDENT UPTAKE AND TOXICITY OF ACIDIC AND BASIC

4

PHARMACEUTICALS IN ZEBRAFISH EMBRYOS (DANIO RERIO)

5 6 7

Lisa Bittnera, Nils Klüvera, Luise Hennebergera, Marie Mühlenbrinka, Christiane Zarflb, Beate

8

I Eschera,b*,

9 10

aHelmholtz

11

bEberhard

12

72074 Tübingen, Germany

Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig

Karls University of Tübingen, Center for Applied Geoscience, Hölderlinstr.12,

13 14

*Address correspondence to [email protected]; Ph: +49 341 235 1244

15 16

1 ACS Paragon Plus Environment


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TOC ART

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19

ABSTRACT

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The aim of the current study was to understand and develop models to predict the pH-

21

dependent toxicity of ionizable pharmaceuticals in embryos of the zebrafish Danio rerio. We

22

found a higher uptake and toxicity with increasing neutral fraction of acids (diclofenac,

23

genistein, naproxen, torasemide, and warfarin) and bases (metoprolol and propranolol).

24

Simple mass balance models accounting for the partitioning to lipids and proteins in the

25

zebrafish embryo were found to be suitable to predict the bioconcentration after 96 h of

26

exposure if pH-values did not differ much from the internal pH of 7.55. For other pH-values,

27

a kinetic ion-trap model for the zebrafish embryo explained the pH-dependence of biouptake

28

and toxicity. The total internal lethal concentrations killing 50% of the zebrafish embryos

29

(ILC50) were calculated from the measured BCF and LC50. The resulting ILC50 were

30

independent of external pH. Critical membrane concentrations were deduced by an internal

31

mass balance model and apart from diclofenac, whose specific toxicity in fish had already

32

been established, all pharmaceuticals were confirmed to act as baseline toxicants in zebrafish.


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33

KEYWORDS

34

zebrafish, pharmaceuticals, internal distribution modelling, aquatic toxicity, pH dependence

35

INTRODUCTION

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According to a recent study more than 40% of the chemicals registered in European

37

chemicals’ legislation REACH1 and approximately 80% of active pharmaceutical ingredients2

38

belong to the group of ionizable organic compounds and are as such able to change their

39

speciation depending on the pH of the surrounding medium. Especially the speciation of weak

40

electrolytes with acidity constants in the range of 4 to 10 need to be considered in

41

environmental risk assessment because these pharmaceuticals will change their speciation at

42

environmentally relevant pH values. Speciation of a pharmaceutical influences its toxicity as

43

demonstrated in several experimental studies that were reviewed by Rendal et al.3 with

44

additional studies published thereafter.4-9

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The difference in toxicity between different chemical species of the same molecule

46

can be explained by differences in cellular uptake and affinity to biomolecules of neutral and

47

ionic species. Both active (carrier-mediated) transport and passive diffusion are possible for

48

membrane permeation.10 In case of passive diffusion, while the neutral fraction can penetrate

49

biomembranes without a significant electrostatic barrier to overcome, the diffusion of ionic

50

compounds is impeded by the membrane dipole.11 Anions showed hindered diffusive

51

membrane permeation as compared to their corresponding neutral species.12 A lower diffusion

52

is expected for organic cations compared to organic anions of similar shape due to higher

53

energy barrier for cations.11 Nevertheless, both, weak organic acids and bases showed

54

substantial membrane permeability through artificial membranes.13 If the charged species

55

cannot cross the membrane or permeates the membrane much slower than the neutral species,

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one can invoke an ion-trapping model.14, 15 Ion-trapping occurs when the neutral fraction of an

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ionizable compound dominates at external pH, but after entering the cell the compound 3 ACS Paragon Plus Environment


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changes to its ionic state due to the different internal pH. The ionic fraction inside the cell is

59

trapped as it is kinetically hindered from passing through the biomembrane. In such cases the

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compound accumulates within the cell. The most extreme case of the ion-trapping model

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(which we term “full ion-trapping model”) is that the neutral species is assumed to have

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externally the same concentration as in the cytosol and the fraction of the charged species in

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each compartment is determined by the fast acid-based equilibrium in the aqueous phases.

64

Ion-trapping models have been previously applied to predict the algal toxicity of acids16 and

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bases4 but reached limitations when the pH differed several log units from the internal pH in

66

the organism. In reality, it is more likely that the diffusion of the charged species is just

67

slower (“kinetic ion-trapping model”). Kinetic ion-trapping models have not yet been

68

implemented in toxicity studies but applied to describe the uptake kinetics into cells,17

69

bacteria18 and for derivation of generic bioconcentration models for cells and aquatic

70

organisms.19

71

The impact of speciation is not limited to the passage through the biomembrane but

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continues at the internal distribution of pharmaceuticals and the concentration(s) at the target

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site(s). As a consequence, the consideration of total exposure concentrations, whether

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externally or internally, is not sufficient if we want to compare toxicity between biological

75

species with different lipid and protein content. The internal freely dissolved concentrations

76

equilibrate with the different components of the cell according to the associated partition

77

constants. A simple mass-balance model has been proposed recently by Goss and

78

coworkers,20 focusing on the partitioning of organic anions.

79

In the present study, we conducted toxicity experiments in embryos of the zebrafish

80

Danio rerio and simultaneously gathered internal and external concentration profiles after 96

81

h at three different pH-values. We measured the pH-dependent uptake of seven ionizable

82

pharmaceuticals, the acids diclofenac, genistein, naproxen, torasemide and warfarin, and the

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bases metoprolol and propranolol, and compared three different toxicokinetic models: (A) a 4 ACS Paragon Plus Environment

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simple model that assumes that the external aqueous concentration equals the internal aqueous

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concentration, (B) a full ion-trapping model and (C) a kinetic ion-trapping model. All three

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models were combined with a mass balance model, similarly to Goss et al.,20 describing the

87

internal distribution in zebrafish embryos to estimate the total concentration in the zebrafish

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embryo and the concentration in the biological membranes. The predicted membrane

89

concentrations were then compared to critical membrane concentrations in zebrafish

90

embryos21 to evaluate if the observed effects were caused by baseline toxicity or specific

91

modes of action.

92

MODEL AND SCENARIOS

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We break up the model to predict the internal concentration of ionizable chemicals into two

94

steps: from external to internal aqueous concentration and from internal aqueous

95

concentration to total body burden or target site concentration (Figure 1).

96



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Figure 1: Two-step model to describe the uptake and internal distribution of ionizable

98

pharmaceuticals in the zebrafish embryo. Cw – external aqueous concentration of the

99

pharmaceutical; ICj – internal concentration in water (j = w), lipids (j = lip) and proteins (j =

100

protein); Klip/w / Kprotein/w – equilibrium partition constant of the pharmaceutical between lipids

101

or proteins and water.

102 103

Conceptually, the uptake kinetics of the charged species are expected to be slower than

104

that of the neutral species. Two extreme scenarios can be defined: equal uptake kinetics of

105

neutral and charged species (Scenario A, full permeability) or the neutral species as the only

106

species that diffuses across the biomembrane (Scenario B, full ion-trap). The reality most

107

likely lies between these two extreme conditions (Scenario C, kinetic ion-trap) (Figure 1).

108 109

From external to internal aqueous concentration. Scenario A, the full permeability scenario,

110

assumed equal external (Cw) and internal (ICw) aqueous concentrations, inside the zebrafish

111

embryo (eq. 1). This holds for neutral chemicals and if the charged species has the same

112

uptake rate constant as the neutral species.

113

Cw (neutral+ion) = ICw (neutral+ion)

(1)

114

The main equation of the full ion-trapping scenario (scenario B) was that the aqueous

115

concentration of the neutral species is the same in the medium and inside the zebrafish

116

embryo (eq. 2).

117

Cw (neutral) = ICw (neutral)

(2)

118

In the full ion-trapping scenario, we assumed that ions are not permeable and the

119

internal speciation, i.e. the fraction of neutral and charged species is calculated with the

120

Henderson-Hasselbalch equation (eq. 3). The external pH together with the pKa determined

121

the fraction neutral of the neutral species, to which the test organism is exposed, as given by


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eq. 3 for acids and eq. 4 for bases. The sum of neutral and charged species of a monoprotic

123

acid or base must be one (eq. 5).

124

Fraction neutral species for acids: neutral =

125

Fraction neutral species for bases: neutral =

126

neutral + ion = 1

127

Expansion of eq. 2 and insertion of eq. 3 or 4 yielded the total aqueous internal concentrations

128

ICw (eqs. 6 and 7).

129

Acids: ICw = ICw(neutral) + ICw(ion) = ICw(neutral) . (1+10 pHinternal-pKa)

(6)

130

Bases: ICw = ICw(neutral) + ICw(ion) = ICw(neutral) . (1+10 pKa-pHinternal)

(7)

1 + 10

1 (pH - pKa)

(3)

1 (pKa - pH)

(4)

1 + 10

(5)

131

In reality it is likely that the charged species makes a contribution to uptake beyond

132

the full ion-trapping scenario. We termed this scenario kinetic ion-trapping scenario (Scenario

133

C in Figure 1) and invoked for this scenario differences in permeability between neutral and

134

charged species and applied the Nernst-Planck equation for the flux of ions across

135

membranes. Such models were already developed for the uptake into cells18, 19 and were in the

136

following adapted for the zebrafish embryo.

137

The uptake rate constant kuptake (eq. 8, in units of m s-1) from the external aqueous into

138

the internal aqueous phase of the zebrafish embryo and the release rate constant krelease (eq. 9,

139

in units of m s-1) were defined in analogy to the model of Zarfl et al.18

140

kuptake = Pn ∙ γexternal,neutral ∙ αexternal,neutral + Pion ∙ eN - 1 ∙ γexternal,ion ∙ αexternal,ion

141 142 143

N

(8) N

krelease = Pn ∙ γinternal,neutral ∙ αinternal,neutral + Pion ∙ eN - 1 ∙ γinternal,ion ∙ αinternal,ion ∙ eN (9)

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In eq. 8 and 9, Pn refers to the permeability of the neutral species and Pion to the permeability

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of the charged species. 7 ACS Paragon Plus Environment


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The Nernst-Planck equation was used to describe the motion of the ionic species

147

across the membrane.18, 19 N is defined by eq. 10 with z = -1 for monoprotic acids and z = +1

148

for monoprotic bases, the universal gas constant R = 8.314 J mol-1 K-1, the Faraday constant F

149

of 96485 C mol-1 and the temperature T of a zebrafish embryo assay is 28°C or 301.15 K. The

150

membrane potential E of the zebrafish embryo membrane is not known, we assumed -0.11 V

151

as for the E. coli.18, 19

152

N=

z∙E∙F R∙T

153

(10)

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The activity coefficient of the neutral species n can be approximated by the Setchenov

155

equation (eq. 11, where I is the ionic strength, Iexternal = 0.05 M, Iinternal = 0.3 M for the typical

156

zebrafish embryo experiment) and the activity coefficient of the charged species ion was

157

calculated by the Davies approximation (eq. 12), where z is the charge (-1 for monoprotic

158

acids and +1 for monoprotic bases).

159

γneutral = 100.3 ∙ I

160 161

(11) 0.5 ∙ |z| ∙

γion = 10

162

(

I 1+ I

- 0.3 ∙ I

)

(12)

163

The ratio between internal ICw and external Cw aqueous concentration at steady state is the

164

ratio kuptake/krelease (eq. 13), which can be simplified because literature evidence suggests that

165

Pion = 10-3.5.Pn.18, 19

166

ICw Cw

kuptake

= krelease =

external,neutral ∙ αexternal, neutral + 10 internal,neutral ∙ αinternal, neutral + 10

-3.5

-3.5

∙

∙

N eN - 1

N eN - 1

∙ external,ion ∙ αexternal,ion

∙ internal,ion ∙ αinternal,ion ∙ eN

(13)

167 168

From internal aqueous concentration to total internal concentration. As a next step, we

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applied a mass balance model to the ICw predicted by all three uptake scenarios to estimate 8 ACS Paragon Plus Environment

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the concentration in the entire zebrafish embryo. For very hydrophilic chemicals such as the

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sulfonamides investigated by Zarfl et al.18 one can assume that the total internal concentration

172

in the cell or organism was equal to the aqueous concentration in the cell or organism. For

173

more hydrophobic IOCs and those that show a strong binding to proteins and lipids, we have

174

to account for the distribution between the different internal compartments of the zebrafish.

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The main sorptive phases in the zebrafish embryo are lipids (lip) and proteins (protein). For

176

simplicity, we assumed that the remaining materials are non-sorptive and behave as water

177

(w).

178

For each species i the fraction in the internal aqueous phase of the zebrafish embryo

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fw,internal(i) is defined by eq. 14, where i can be HA, A-, B, BH+ or any other species.

180

fw,internal(i) =

1 1 + Klip/w (i) ∙

Vflip Vfw

+ K

protein/w

(i) ∙

Vfprotein Vfw

(14)

181

Klip/w(i) refers to the lipid-water partition constant of species i, Kprotein/w(i) is the protein-water

182

partition constant of species i. This internal mass balance model considers protein binding as

183

a partitioning process. Vf refers to the volume fraction of the different phases, i.e. lipids

184

(Vflip), proteins (Vfprotein) and water (Vfw).

185

The total internal concentration of species i, IC(i), was then calculated according to eq.

186

15:

187

IC(i) = ICw(i) fw,internal(i)

188

If several species were involved (i = neutral, anion, cation), then the partition constant of one

189

species was replaced by the pH-dependent distribution ratio of all species Dlip/w(pH) (eq. 16)

190

and Dprotein/w(pH) (eq. 16).

191

Dlip/w(pH) = ∑i = 1αi × Klip/w(i)

192

Vfw

(15)

n

(16)



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i were calculated with eqs. 3 and 4 for monoprotic acids and bases. The internal pH in the

194

zebrafish embryo is 7.55.22 Eq. 14 could then be transformed to eq. 17.

195

fw,internal =

1 1 + Dlip/w(pH 7.55)

Vflip Vfw

+ Dprotein/w(pH7.55)

Vfprotein Vfw

(17)

196

The total lipid content of the zebrafish embryo is 4.54 glip/Lembryo8 and the total protein

197

content is 46 gprotein/Lembryo.23 The total volume of zebrafish embryos 96 hour post fertilization

198

(hpf) is approximately 440 nL.24 Hence one zebrafish embryo contains approximately 2 µg

199

lipid and 20 µg protein. For simplicity, we assumed a density of 1 kg/L for all phases. The

200

protein and lipid content is remarkably constant over the entire exposure duration from

201

fertilization to 96 hpf because the zebrafish embryo feeds only from its yolk sac and not from

202

the outside.23 Hence we define Vflip= 0.00454 Llip/Lembryo, Vfprotein = 0.0455 Lprotein/Lembryo and

203

Vfw = 0.94996 Lw/Lembryo.

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The lipid phase is made up of storage and membrane lipids. Since the energy storage in the

205

zebrafish egg is the yolk, which is rich of phospholipids,23 we used membrane lipids as

206

surrogate for lipids, namely the liposome-water distribution ratio Dlip/w(pH).

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The protein-water distribution ratio Dprotein/w(pH) was approximated by the distribution

208

ratio to bovine serum albumin (BSA) DBSA/w(pH) or to structural or muscle proteins (MP)

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DMP/w(pH). Especially organic anions have a high affinity to BSA and specific binding sites25.

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Thus, BSA is not the perfect surrogate for zebrafish embryo proteins, which are composed

211

mainly of the yolk protein vitellogenin.26 Since there were no experimental data available for

212

Dvitellogenin/w(pH), we used MP as surrogate for the proteins in the zebrafish embryo.

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We invoked a modification of eq. 13 to describe the relationship between the aqueous internal

214

concentration ICw and the total internal concentration IC in the organism (eq. 18).

215 216

IC =

Cw,internal

∙ Vfw

fw,internal

= ICw(Vflip × Dlip/w(pH 7.55) + Vfprotein × Dprotein/w(pH 7.55) + Vfw)

(18)


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Bioconcentration factor. The bioconcentration factor (BCF) is defined as the ratio of total

218

internal, IC, and aqueous external concentration (Cw) at steady state (eq. 19).

219

BCF = Cw

220

We were not able to determine uptake kinetics but previous work24 has demonstrated that

221

during the 96 hours of the standard zebrafish embryo toxicity test a steady state is reached for

222

many chemicals. Hence, we can compare the experimentally determined BCF96hpf (96 hours

223

post fertilization, of which approx. 94h was the chemical exposure duration, see Methods for

224

more details) with the predicted BCF from the three scenarios. Scenario A assumed that both

225

neutral and charged species are equally fast membrane-permeable, hence no ion-trapping was

226

invoked and the associated BCF is termed BCFMB (with MB referring to mass balance). Then

227

ICw = Cw and eq. 19 simplified to eq. 20 for both, acids and bases.

228

BCFMB(pH) = fw,internal

IC

(19)

1

(20)

229

Inserting eqs. 6 and 7 (scenario B) into the basic equation for the BCF (eq. 19) one

230

obtains eqs. 21 and 22 describing the BCFfull ion-trap, which are just dependent on pH, pKa, Vfw

231

of the organism and the substance fraction in the internal aqueous phase fw,internal(i):

232

Bases: BCFfull ion - trap(pH) =

IC(neutral) + IC(ion, pHinternal) Cw(neutral) + Cw(ion, pHexternal)

=

IC(neutral) + IC(ion, pHinternal)

1 w,internal(neutral)

Vfw(f

1 + 10 Vfw (f

+

pKa - pHinternal 10 fw,internal(ion) )

pKa - pHexternal

1

w,internal(neutral)

+

pH - pKa 10 internal fw,internal(ion) )

(21)

233

Acids: BCFfull ion - trap(pH) =

234

To relate the kinetic ion-trapping scenario (scenario C) to the BCF, we need to insert eq. 13

235

into eqs. 18 and 19 yielding eq. 23:

236 237

external,neutral ∙ αexternal, neutral + 10

BCFkinetic ion - trap(pH) =

=

Cw(neutral) + Cw(ion, pHexternal)

internal,neutral ∙ αinternal, neutral + 10

-3.5

-3.5

∙

∙

1 + 10

N eN - 1

N eN - 1

pHexternal - pKa

∙ external,ion ∙ αexternal,ion N

∙ internal,ion ∙ αinternal,ion ∙ e

(22)

Vfw

∙ fw,internal

(23)

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Internal lethal concentration ILC50 and internal lethal membrane concentration ILC50lip.

240

The internal lethal concentration was calculated from BCF and LC50 (eq. 24) for all three

241

scenarios.

242

ILC50 = BCF . LC50

243

For hydrophobic chemicals, a lipid normalization is often made to account for the uptake into

244

biological membranes. For ionizable and more polar chemicals, a full mass balance must be

245

derived instead, because binding to proteins might become very important and also the

246

fraction in the aqueous phase is not negligible. The fraction in the lipids flip,internal was defined

247

analogously to fw,internal by eq. 25.

248

flip,internal =

249

(24)

1 1+

1 Dlip/w(pH 7.55)

∙

Vfw Vflip

+

Dprotein/w(pH 7.55) Dlip/w(pH 7.55)

∙

Vfprotein Vflip

(25)

The internal membrane concentration causing 50% of lethality, ILC50lip is then

250

defined by eq. 26.

251

ILC50lip =

252

Alternatively, ILC50lip can also be calculated by multiplication of the ICw by Dlipw(pH 7.55).

253

The critical membrane concentration of baseline toxicity for the 96h zebrafish embryo

254

toxicity test was shown to be 226 mmol/kglipid,21 independent of considering neutral or

255

charged chemicals.21

256

METHODS

257

Chemicals. Test chemicals were purchased in highest available purity as diclofenac sodium

258

(CAS 15307-79-6, purity ≥99%, Cayman Chemical Company), (S)-naproxen (CAS 22204-

259

53-1, purity ≥99%, Cayman Chemical Company), genistein (CAS 446-72-0, purity ≥98%,

260

Cayman Chemical Company), warfarin (CAS 81-81-2, purity ≥98%, Fluka), torasemide (CAS

261

56211-40-6, purity ≥98%, Sigma-Aldrich), metoprolol tartrate (CAS 56392-17-7, purity

262

≥98%, Sigma-Aldrich) and propranolol hydrochloride (CAS 318-98-9, purity ≥99%, Sigma-

flip,internal ∙ ILC50

(26)

Vflip


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263

Aldrich). All concentrations were expressed as molar concentrations related to the free acid

264

and base, neglecting potential toxicity of the counterions of the salt. The physicochemical

265

properties relevant for further calculations are summarized in Table 1. Partition constants

266

between bovine serum albumin (BSA) and water, KBSA/w that were only experimentally

267

available for acids measured at a pH with a fraction of neutral species HA >0.01 were back-

268

calculated to their ionic contribution with eq. 27.

269

KBSA/w(ion) =

270

Partition constants were not abundant for muscle proteins (KMP/w). The KMP/w(neutral) were all

271

predicted with a poly-parameter linear free energy relationship (pp-LFER) taken from the

272

freely accessible pp-LFER Database.27 Due to a lack of reliable prediction models the

273

KMP/w(ion) were deduced from the difference between logKMP/w(neutral) and KMP/w(ion) for

274

those three chemicals (diclofenac, naproxen, and propranolol) where experimental KMP/w(ion)

275

were available.28 We used the logKMP/w(neutral)- logKMP/w(ion) of 1.04, which is the mean of

276

the values of diclofenac and naproxen for all acids and logKMP/w(neutral)- logKMP/w(ion) of

277

0.76 for the bases.

DBSA/w(pH 7.4) - αHAKBSA/w(neutral) 1 - αHA

(27)

278



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279

Table 1: Physicochemical parameters of the test set of acids and bases, including the acidity constant pKa, the partition constant between membrane

280

lipids (liposomes) and water, logKlip/w, the partition constant between bovine serum albumin (BSA) and water, logKBSA/w, and the partition constant

281

between muscle protein and water, logKMP/w of the neutral and ionic species (anion for acids, cations for bases). Pharmaceutical

pKa

logKlip/w

logKlip/w

logKBSA/w

logKMP/w

logKMP/w

(neutral)

(ion)

(neutral)

(neutral)

(ion)

4.45a

2.64a

3.87b

4.40c

3.27b

2.47d

3.65f

3.30g

2.68b

2.90h

2.08b

1.04s

logKBSA/w (ion)

Acids 3.99a

Diclofenac Genistein

7.20, 10.0, 13.1e

Naproxen

4.18i

3.84f

1.92g

3.37b

5.21c

2.61b

1.33d

Torasemide

6.68j

3.48f

2.23g

2.86b

3.88k

2.54b

1.50s

Warfarin

4.90l

3.39l

1.40l

2.04b

3.46c

1.74b

0.70s

Metoprolol

9.68m

1.27n

1.43o

1.10b

1.51p

0.87b

583

-

Torasemide

5.5

0.586±0.042

>37.8

-

Warfarin

5.5

10.6±0.7

88.8

18.5

7.0

0.744±0.080

>76.3

-

7.0

2.68±0.49

19148

113

8.0

12.4±0.4

1748

47.4

8.6

52.0±4.1

53.88

62.0

5.5

2.33±0.19

24178

550

7.0

33.3±4.6

1288

417

8.0

234±28

22.88

526

Naproxen

Bases Metoprolol

Propranolol

387 388

The measured BCF96hpf for diclofenac in the zebrafish embryos were in the same range

389

as the steady-state experimental BCF of 3-5 L L-1 in rainbow trout at pH 7.4-8.4

390

96h-BCF in Galaxias maculatus of 87 at a pH of 6.7

391

again the expected pH-dependency.

43

42

while the

was substantially higher, reflecting

392 393

Modelling partition constants between zebrafish and water. Experimentally determined

394

BCF96hpf (Table 2) were compared with the modelled BCF for the different model scenarios

395

(Figure 2). For all pharmaceuticals, the mass balance model underperformed as it could not

396

reflect the pH-dependency of the experimental BCF96hpf. The two ion-trapping models came

397

to very similar predictions of BCF for genistein, metoprolol and propranolol and in most

398

cases (with exception of diclofenac at pH 5.5, naproxen at pH 8.0, torasemide at pH 5.5 and

399

propranolol at pH 7 and 8) the model outputs of these two models reflected the experimental

400

data within a factor of 10 (Figure 2). 20 ACS Paragon Plus Environment

Page 21 of 35


401 402

Figure 2: Comparison of modelled BCFMB, BCFfull

403

experimentally determined BCF96hpf for pharmaceuticals: A. diclofenac, B. genistein, C.

404

naproxen, D. torasemide, E. warfarin, F. metoprolol and G. propranolol. The solid line is the

405

1:1 line (full agreement of experiment and model), the dotted lines mark the deviation by a

406

factor of 10 in both directions.

ion-trap

and BCFkinetic

ion-trap

and

407 408

The kinetic ion-trap and the full ion-trap models gave very similar predictions when

409

the external pH was within two to three log units from the pKa, which was the case for

410

genistein, torasemide, metoprolol and propranol (Figure 2B, D, F,G). For the carboxylic acids

411

diclofenac (pKa 3.99) and naproxen (pKa 4.18) where the pH-pKa difference was larger, there

412

was a substantial difference between BCFfull ion-trap and BCFkinetic ion-trap (Figure 2A and C) and

413

a minor difference for warfarin (Figure 2E). In case of diclofenac the BCFkinetic

414

performed better and for naproxen the BCFfull ion-trap was closer to the experimental BCF96hpf

415

(Figure 2A).

ion-trap

416

All applied models assumed equilibrium conditions for their application, but

417

especially in cases where ionic species dominated in external solution the uptake across the 21 ACS Paragon Plus Environment


418

lipid bilayer membrane could be significantly reduced. As we did not measure time-

419

dependent uptake in the present study we cannot proof that steady state had been reached.

420

Brox and coworkers24 studied the uptake of selected ionizable compounds in the zebrafish.

421

They measured equilibrium conditions after 72 h for most of their test compounds including

422

metoprolol at pH 7.4. However, some of their compounds, including clofibric acid, which has

423

similar physicochemical properties as the carboxylic acids investigated here, did not reach

424

steady state or even decreased in concentration. As main reasons metabolism and affinity to

425

efflux transporters were discussed, which are processes that are not included in our present

426

modelling approach due to the lack of experimental data appropriate to parameterize extended

427

models. Measured internal concentrations showed a high variance especially in the presence

428

of mostly ionic species. This could be an indication that equilibrium was not reached within

429

94 h exposure time and uptake was influenced by individuals’ movement and hatching.

430

How well the BCF model performs is not only determined by the uptake model but

431

also by the internal distribution in the embryos. The relative slope against the slope 1 of the

432

1:1 line (solid line in Figure 2) is a measure of the performance of the uptake model and the

433

absolute deviation from the 1:1 line rather reflects the performance of the internal distribution

434

model, which is largely driven by the partition constants. The main input parameters of the

435

internal distribution model are the compartment sizes, i.e., the protein, lipid and water content

436

of the embryo, and the partition constants. As there is no consistent deviation in one direction

437

for all pharmaceuticals, we can safely assume that the measurements of protein and lipid

438

content are realistic, which is also expected because the measurements were performed in the

439

same laboratory (UFZ) that has kept robust populations over many years.23

440

For estimating the internal distribution, reliable partition constants are needed.

441

Especially the availability of partition constants to structural proteins is limited to few

442

experimental measurements.28,

443

database27 the data situation has improved a lot but these easy-to-use models are limited to 22

44

By the introduction of prediction tools like the pp-LFER

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Page 22 of 35

Page 23 of 35


444

neutral chemicals. There exist promising approaches for the prediction of Klip/w45 and KBSA/w

445

for ions46, 47 but in our model the main driver is the Kprotein/w with proteins not well represented

446

by BSA but rather by vitellogenin. To our knowledge there are no Kvitellogenin/w available in the

447

literature, at least not for the pharmaceuticals tested here. In absence of Kvitellogenin/w we relied

448

on muscle protein as surrogate, but all logKMP/w of neutral species had to be predicted with the

449

pp-LFER database27 and experimental values for logKMP/w(ion) were only available for

450

diclofenac, naproxen and propranolol. Hence, model uncertainty could be best reduced by

451

future determination of Kvitellogenin/w for neutral and charged species.

452 453

pH-Dependent effects. We detected effects on the survival of zebrafish embryos for four of

454

the five acids (Figure 3). Concentration-response curves are included in the SI, Figure S8 and

455

the derived LC50 are listed in Table 2. For torasemide no toxicity was detected up to the

456

highest measured exposure concentration of 37.8 µM already at pH 5.5. The % lethality of

457

diclofenac, genistein and naproxen at pH 8.0 was not sufficiently high to model

458

concentration-response curves and to derive a LC50, up to the highest test concentrations of

459

109 µM, 40.5 µM and 583 µM respectively. For warfarin no LC50 could be derived at pH 7.0

460

up to the highest test concentration of 76.3µM. The pH-dependent LC50 values of the bases

461

metoprolol and propranolol from a previous study8 were also included in Table 2. All acidic

462

pharmaceuticals showed a decreasing LC50 value with decreasing pH and increasing neutral

463

fraction (Figure 3).



A. Diclofenac

B. Genistein

1 4

6

8 pH

10

0.0 12

1.5

0.5

1.0 0.5 0.0

4

10

log(1/LC50) [mM]

log(1/LC50) [mM]

neutral

464

3 2

0.5

1 0

4

0.0 12

1

cation

0.5

4

6

8 pH

anion

10

10

2

-1 -2

8 pH

0.0 12

F. Propranolol 1.0

0

6

0.0 12

di-anion

1.0

1

fraction

8 pH

0.0 12

fraction

6

fraction

0.5 0.5

4

10

2

1.0

1.0

0.0

8 pH

1.0

E. Metoprolol

D. Warfarin 1.5

6

log(1/LC50) [mM]

0

2.0

log(1/LC50) [mM]

0.5

4

fraction

2

C. Naproxen

1.0

fraction

3

2.5

log(1/LC50) [mM]

1.0

fraction

log(1/LC50) [mM]

4

Page 24 of 35

0

0.5

-1 -2

4

6

8 pH

10

0.0 12

tri-anion

465

Figure 3: LC50 values (left y-axis) of pharmaceuticals in zebrafish embryos at 96 hpf and

466

speciation (right y-axis) plotted against the pH. The fraction i of species i (i = neutral, cation,

467

anion, di-anion, tri-anion) was calculated according to eq. 3 and 4 and for genistein taken

468

from reference7; A. diclofenac, B. genistein, C. naproxen, D. warfarin, E. metoprolol8 and F.

469

propranolol.8

470 471

LC50 data for diclofenac, genistein, naproxen and warfarin in zebrafish embryos are

472

available in literature. Especially diclofenac has received much attention in recent years as it

473

leads to histopathological changes in fish48 and is suspected to have effects on neural

474

development.49 The other nonsteroidal anti-inflammatory drugs as well as the -blockers have

475

also received ample attention in the literature.8 Despite the availability of toxicity data, their

476

quality suffers from insufficient data documentation, especially regarding pH-conditions of

477

exposure. We therefore assumed the pH-range recommended by the OECD guideline 236 of

478

pH 6.5 to 8.5 with allowed maximum fluctuation of 1.5 pH-units during the 96 h of 24 ACS Paragon Plus Environment

Page 25 of 35


479

exposure.40 Taking this into account our results generally agreed with published LC50 data

480

(SI, Table S16). A difference larger than a factor of two was only detected for naproxen, with

481

a LC50 of 500 µM published by Li et al.50 compared to LC50 of 10.7 µM at pH 5.5 and 188

482

µM at pH 7.0 in the present study.

483

The experimental LC50 for diclofenac were 0.811 µM at pH 5.5, 12.1 µM at pH 7 and

484

123 µM at pH 8. While the LC50 of 24.5 µM (7.8 mg/L) for diclofenac published by van den

485

Brandhof et al.51 with exposure until 72 hpf, at pH 8.07 is in line with LC50 of 12.1 µM (3.85

486

mg/L) published by Praskova et al.,52 no mortality was described in other studies at higher

487

concentrations up to 39 µM (12.5 mg/L).53 In another study, a mortality of 68 % was detected

488

with exposure of 16 µM diclofenac (5.19 mg/L, 0-96 hpf, pH 7.0-7.2).42 Given the strong pH

489

dependence in our experiments, we can conclude that the literature data are within the

490

expected range of effects.

491

Experimental LC50 values of genistein were 10.1 µM and at pH 5.5 and 8.84 µM at

492

pH 7.0, which are similar to the LC50 of 16.3 reported by Sarasquete et al.54 For warfarin we

493

derived a LC50 of 88.8 µM at pH 5.5 but were not able to detect any toxicity at pH 7.0. We

494

were not able to reach a concentration range even close to the LC50 of 988 µM reported in

495

literature55 due to the limited solubility even in the anionic state. An LC50 of 988µM at pH 7

496

would lead to an ILC50membrane close to baseline toxicity. For torasemide we experienced a

497

similar problem, as the concentration range tested in the present study was limited by the poor

498

water solubility of this pharmaceutical and we were not able to reach an aqueous

499

concentration even close to baseline toxicity. Given the measured BCFhpf of 0.59 of

500

torasemide an external exposure concentration of 4950 µM would have been required to reach

501

the ILC50 baseline toxicity of 226 mmol/kg lipid.21

502 503

Critical membrane concentrations of baseline toxicity. The ILC50lip that was calculated from

504

experimental LC50 and experimental BCF96hpf remained virtually unaffected by the external 25 ACS Paragon Plus Environment


Page 26 of 35

505

pH, which confirms our earlier work on -blockers8 and antihistamines.9 We compared the

506

ILC50lip to the ILC50baseline

507

Klüver and coworkers.21 As is shown in Figure 4A, all pharmaceuticals were in the range of

508

baseline toxicity (23 mmol/kglipid < ILC50baseline

509

diclofenac that was more toxic with ILC50lip of 1.91 – 5.13 mmol/kglip. Diclofenac is a non-

510

steroidal anti-inflammatory drug (NSAID) and has been extensively studied in zebrafish

511

embryos. Special emphasis was put on its neurotoxic effects,49 but also effects on the

512

cardiovascular system are reported in addition to several effects on phenotype.56 On the gene

513

level diclofenac is known to interfere with a variety of pathways, including metabolic activity,

514

cell cycle mechanisms, hormone production and others.57 As diclofenac acts on these many

515

targets it is not possible to specify only one mode of action. The multiple target sites might

516

even add up to cause the elevated toxicity we observed.

toxicity

of 226 mmol/kglipid for zebrafish embryos published by

ILC50 [mmol/ kglip]

ILC50lip [mmol/ kglip]

104 103 102 101 100 4

6

8 pH

517

< 2260 mmol/kglipid) except for

B. Kinetic ion-trap model

A. Experimental

10-1

toxicity

10

104

Diclofenac

103

Genistein

102

Naproxen

101

Warfarin

100

Metoprolol

10-1

Propranolol 4

6

8

10

pH

518

Figure 4: ILC50lip for all pharmaceuticals detected with lethal effects based on A.

519

experimentally determined BCF96hpf and B. BCFkinetic ion-trap. ILC50baseline toxicity was taken from

520

reference21 with 226 mmol/ kglipid, dotted lines represent the factor-of-10 range of baseline

521

toxicity.

522


Page 27 of 35


523

When applying the kinetic ion-trap model, the ILC50lip values remained in the range

524

of baseline toxicity, except for naproxen and metoprolol. Diclofenac remained classified as

525

specifically acting but the difference between the baseline toxicity and the more toxic

526

diclofenac was less pronounced. In absence of experimental data on internal effect

527

concentrations and/or bioconcentration factors, the kinetic ion-trap model may serve to

528

estimate critical membrane concentrations to allow one to differentiate whether chemicals are

529

baseline toxicants or specifically acting.

530

Previous ion-trapping models have modelled effects as a function of external

531

concentration. We broke up such models into two steps, one accounting for the uptake into

532

the aqueous phase inside the embryo and the second for the internal distribution in the

533

embryo. We were able to validate this approach by measuring internal concentrations in the

534

fish embryos. When evaluating the three model approaches, a simple mass balance, full ion-

535

trapping and kinetic ion-trapping, using partition constants as Klip/w and KMP/w, we achieved

536

the best fit to our experimental data with the kinetic and full ion-trapping models combined

537

with the internal distribution model.

538

The kinetic ion-trapping models should be parameterized better with respect to

539

embryo-specific parameters such as the membrane permeability ratios Pn/Pion and the

540

membrane potential of the zebrafish membranes. Simple mass balance models will perform

541

sufficiently good when dealing with neutral compounds and at pH values that are similar to

542

the internal pH of zebrafish embryos of 7.55.22

543

544

ACKNOWLEDGEMENT

545

The research leading to these results has received support from the Innovative Medicines

546

Initiative Joint Undertaking under iPiE grant agreement n° 115735, resources of which are

547

composed of financial contribution from the European Union's Seventh Framework 27 ACS Paragon Plus Environment


Page 28 of 35

548

Programme (FP7/2007-2013) and EFPIA companies’ in-kind contribution. Funding by the

549

Excellence Initiative of the German Federal Ministry of Education and Research (BMBF) and

550

the German Research Foundation (DFG) at the University of Tübingen is gratefully

551

acknowledged. The authors’ special thanks goes to Niklas Wojtysiak for his extraordinary

552

help with the toxicity experiments. We are grateful towards Alison Nimrod Perkins and Anja

553

Coors for helpful discussions.

554

SUPPORTING INFORMATION

555

The Supporting Information contains additional information on the extractions, chemical

556

analysis, the linear regressions for derivation of the BCF and all concentration-response

557

curves and is available at …..

558

DISCLAIMER

559

All authors have no interest to declare.

560

561

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Combined ion-trapping and mass balance models to describe the pH

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