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Toxic mixtures in time – the sequence makes the poison Roman Ashauer, Isabel O'Connor, and Beate I. Escher Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b06163 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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Toxic mixtures in time – the sequence makes the poison

1 2

Roman Ashauer1,2*, Isabel O’Connor1, Beate I. Escher1,3,4

3 4 5 6 7 8 9 10 11 12

1

Department of Environmental Toxicology, Eawag - Swiss Federal Institute of Aquatic

Science and Technology, 8600 Dübendorf, Switzerland 2

Environment Department, University of York, Heslington, York YO10 5DD, United

Kingdom 3

Cell Toxicology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany

4

Environmental Toxicology, Center for Applied Geosciences, Eberhard Karls University

Tübingen, Germany

13 14 15 16

*Corresponding author:

17

Phone: +44-(0)-1904-324052

18

Email: [email protected].

19 20 21

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Abstract: “The dose makes the poison.” This principle assumes that once a chemical is

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cleared out of the organism (toxicokinetic recovery), it no longer has any effect. However, it

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overlooks the other process of re-establishing homeostasis, toxicodynamic recovery, which

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can be fast or slow depending on the chemical. Therefore, when organisms are exposed to

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two toxicants in sequence, the toxicity can differ if their order is reversed. We test this

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hypothesis with the freshwater crustacean Gammarus pulex and four toxicants that act on

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different targets (diazinon, propiconazole, 4,6-dinitro-o-cresol, 4-nitrobenzyl chloride). We

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found clearly different toxicity when the exposure order of two toxicants was reversed, while

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maintaining the same dose. Slow toxicodynamic recovery caused carry-over toxicity in

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subsequent exposures, thereby resulting in a sequence effect – but only when toxicodynamic

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recovery was slow relative to the interval between exposures. This suggests that carry-over

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toxicity is a useful proxy for organism fitness and that risk assessment methods should be

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revised as they currently could underestimate risk. We provide the first evidence that carry-

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over toxicity occurs amongst chemicals acting on different targets and when exposure is

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several days apart. It is therefore not only the dose that makes the poison but also the

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exposure sequence.

38 39 40

Keywords: Toxicology, dose, mixture toxicity, chemical safety assessment

41



42

Introduction

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The paradigm “the dose makes the poison”, that is, that chemicals can be toxic or non-toxic

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depending on their dose, stems back to Paracelsus. Given that exposure to toxicants in the

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environment is often episodic and repeated 1, 2 and organisms are exposed to several toxicants

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at the same time or after each other, it is important to also take into consideration the

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temporal aspects of toxicity 3-5. The time required by an organism to recover from damage

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due to previous exposure events is defined as the organism recovery time 6, 7. Organism

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recovery results from toxicokinetic recovery, i.e. biotransformation, distribution and

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excretion of the toxicant, and toxicodynamic recovery, i.e. re-establishment of homeostasis.

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In previous studies 7-9, we developed toxicokinetic-toxicodynamic models using toxicants

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from different chemical classes and found that certain toxicants exhibit slow toxicodynamic

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recovery in Gammarus pulex. The modelling suggested that even after the substance was

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eliminated from the organism, more time was necessary to re-establish homeostasis. The

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conventional wisdom, as reflected by most current chemical risk assessment practices10, is

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that when a chemical is eliminated from an organism (toxicokinetic recovery), it no longer

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exerts an effect. This overlooks a subtler process of toxicodynamic recovery, which is

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determined by the mode of toxic action. Both aspects of recovery are related to the

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characteristics of the substance 3, 9, where toxicokinetic parameters are related to

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hydrophobicity 11 and toxicodynamic parameters to mechanism of toxicity 9 and hence

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molecular structure 12.

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What if subsequent exposure events occur in intervals that are sufficient for

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toxicokinetic recovery but not for toxicodynamic recovery? We already know that

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toxicodynamic build-up of damage can lead to carry-over toxicity from subsequent pulses of

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the same toxicant 7 and from subsequent pulses of different toxicants that act on the same

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target 6. Therefore toxicokinetic-toxicodynamic models were developed recently, that extend

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the classical dose paradigm by complementing internal concentrations with scaled damage as

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the dose metric 9, 13. These early insights into toxicokinetics and toxicodynamics led to the

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sequence effect hypothesis: when organisms are exposed to two toxicants in sequence, the

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toxic effects may differ if that sequence is reversed 6.

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To date this hypothesis has only been tested once, with carbaryl and chlorpyrifos in

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G.pulex, two toxicants that both inhibit the same enzyme. The empirical evidence supported

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it but it could be explained by a build-up of inhibited enzyme 6 and therefore did not

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challenge the current paradigm. More recently we gained a much deeper understanding of

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toxicokinetics 14 and toxicodynamics 9 of a diverse range of toxicants in G. pulex, in ACS Paragon3Plus Environment

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particular how toxicokinetic and toxicodynamic recovery times differ among chemicals.

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Based on this new knowledge we now propose a significant broadening of the sequence

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effect hypothesis: it also applies to toxicants that act on different targets and via different

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mechanisms. Would carry-over toxicity also occur after subsequent pulses of different

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toxicants that act on different targets with different toxic modes of action? This is an

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important question because sequential exposure to toxicants is the norm, but the current risk

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assessment paradigm pays little attention to possible interactions of different toxicants over

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time. We think that substances acting on different targets can cause carry-over toxicity, which

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we view as a proxy for reduced organism fitness. If the sequence effect occurs for a wide

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range of toxicants with different mechanisms of toxicity, then we are potentially severely

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underestimating risk from chemicals.

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The sequence effect hypothesis can be tested experimentally because it predicts that

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the toxicity of two subsequent pulsed exposure events is more pronounced if we first expose

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organisms to the substance with a slower recovery time, followed by exposure to the

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substance with a faster recovery time. Conversely, exposure to the substance with a shorter

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recovery time followed by the substance with a longer recovery time will result in less carry-

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over toxicity and have a weaker impact on the organism.

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We tested the sequence effect hypothesis in the amphipod crustacean Gammarus

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pulex using four model toxicants that differ in their toxic mode of action. G.pulex are

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particularly suitable for this type of study because of their relatively long life-span and

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sufficiently large size to measure toxicant body residues. Diazinon [DIAZ] is an insecticide

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that inhibits acetylcholinesterase 15, propiconazole [PCZ] is a fungicide that likely acts as a

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baseline toxicant in G. pulex 16, 4,6-dinitro-o-cresol [DNOC] is an uncoupler of oxidative

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phosphorylation 17, and 4-nitrobenzyl chloride [NBCl] is a reactive toxicant that forms

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covalent bonds with proteins and other biomolecules 18. Therefore, each toxicant acts

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according to a different mode of action, and their mixture toxicity should be explained by the

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model of independent action 19, 20. Previously, we determined the times required for 95%

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elimination of the toxicologically relevant metabolites and for 95% recovery of

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toxicodynamic damage in G. pulex 7, 8, 14, 21 for each compound individually (Table 1). All

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four substances have toxicokinetic recovery times of less than three days, whereas the

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toxicodynamic recovery times are short for PCZ (< 3d) and DNOC (29000d). Based on this knowledge, we designed four experiments,

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each involving two toxic experimental groups and a control group. The two toxic

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experimental groups employed two subsequent pulsed exposures to two different toxicants ACS Paragon4Plus Environment


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(Fig. 1). The two toxic experimental groups in each experiment differed only in the sequence

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of the toxicants applied.

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We chose a pulse interval that was long enough to allow toxicokinetic recovery (such

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that no residual chemical remained in the body at the onset of the second pulse (Supporting

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Information Fig. S1–S4)), but too short to allow complete toxicodynamic recovery after

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DIAZ and NBCl exposure. Hence, we expected carry-over toxicity for those experimental

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groups in which the first applied pulse was a toxicant with slow toxicodynamic recovery

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(DIAZ or NBCl) but not in the groups in which the first applied pulse was a toxicant with fast

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toxicodynamic recovery (PCZ and DNOC). Consequently, we expected to observe a

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sequence effect, i.e. differences in survival at the end of the experiment, in the first, second

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and third experiments (Fig. 1). In the fourth experiment, we expected both experimental

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groups to exhibit carry-over toxicity due to slow toxicodynamic recovery of both toxicants,

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and therefore did not expect the sequence effect.

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We also carried out toxicokinetic-toxicodynamic mixture toxicity modelling. This

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dynamic modelling expands the applicability domain of mixture toxicity models to pulsed or

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time-variable exposures. The model structure was derived by assuming that the toxicants act

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on different targets and therefore independently. The model parameters were taken from

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previous studies. This enables us to compare model forecasts with new experimental data

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from this study to assess how good the model predictions are.

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Materials and methods

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Experiments

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Experimental procedures closely followed established protocols 6, 7, 9. Adult G. pulex were

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collected in a headwater stream in the Itziker Ried, Switzerland near Zurich (E 702150, N

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2360850) and acclimatized to 13°C (Table S2). G. pulex were fed horse-chestnut leaf discs

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conditioned with Cladiosporum herbarum22 fungi. Experiments were performed in pyrex

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beakers filled with 500 mL pre-aerated artificial pond water22 and at 13°C under a 12:12 h

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light:dark cycle. Each beaker initially contained 10 G. pulex and the total number of test

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organisms in each toxic experimental group is given in Tables S6, S 8, S10 and S12. Test

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organisms were a natural mixture ratio of adult males and females and the numbers used in

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each experiment varied depending on the availability of organisms at the Itziker Ried. Leaf

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discs were added for ad libitum feeding, ranging from three to five leaf discs per beaker

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depending on the number of alive G.pulex. Live organisms were counted by gently prodding

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with a spatula and observing their resulting movement (Tables S6, S8, S10, and S12). Each ACS Paragon5Plus Environment

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experiment included also a control group which was not treated with toxicants. No blinding

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was employed. Any neonates that were released by females during the experiments were not

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counted. Dead individuals were removed.

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The test concentrations were chosen based on previous toxicity studies7-9 to achieve partial

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mortality after the first pulse. The test chemicals were dosed as a mixture of 14C-labelled and

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unlabelled substances to allow quantification of exposure concentrations. Radiolabelled

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DIAZ and PCZ were supplied by the Institute of Isotopes (Budapest, Hungary). Radiolabelled

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DNOC and NBCl were sourced from American Radiolabeled Chemicals (St. Louis, MO,

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USA). Unlabelled chemicals were of analytical grade and supplied by Sigma-Aldrich (Buchs,

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Switzerland). Dosing stocks of labelled and unlabelled test compounds were prepared in

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acetone as solvent. Each beaker was dosed by pipetting the required volume of stock into the

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medium, followed by gentle stirring with a glass rod. Concentrations of the solvent acetone in

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the test medium were 0.1% or lower (Table S4), i.e. three orders of magnitude below

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concentrations toxic to crustaceans23. Actual exposure concentrations were measured by

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liquid scintillation counting (Tri-Carb 2200CA, Packard, USA) using 1 mL aliquots of the

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medium (Tables S5, S7, S9, and S11), correcting for efficiency using internal standards,

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subtracting background activity using control medium samples and converting radioactivity

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to molar concentrations by using the known mixture ratio of labelled and unlabelled

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substance. Immediately before and at the end of an exposure pulse, organisms were separated

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from the medium by using a tea strainer, the test medium was discarded, and the organisms

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were rinsed with clean water and then placed in beakers with fresh artificial pond water and

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fresh leaf discs. The pH, conductivity and dissolved oxygen concentration of the medium

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were monitored (Table S4).

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Our experimental design aimed to maximise the number of test organisms that we could

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handle so that we would maximise experimental power. However no formal power analysis

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was carried out and the experimental design was constrained by the practicalities of the

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experiment.

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Statistical Analysis

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Statistical testing was performed using GraphPad Prism (v6.03, GraphPad Software Inc., La

173

Jolla, CA, USA). To detect the sequence effect, we compared the numbers of dead and live

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individuals at the end of the experimental groups using contingency table analysis, more

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specifically, Fisher’s exact test (Fig. 1B, D, F, H, J, L, N, P). For evaluation of carry-over

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toxicity, we compared the survival curves after the first and second pulses for each



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experimental group using the Gehan-Breslow-Wilcoxon test (Fig. 2). These survival curves

178

were constructed by recording the absolute number of alive G.pulex at the start of a pulse

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(this could be the first or the second pulse in an experimental group) as the initial number of

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organisms and then following each group until either the end of the experiment or the onset

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of the second pulse. In other words: we compared survival curves during and following the

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pulsed exposure to the same toxicant in different groups of the same experiment, once as the

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first pulse and again as the second pulse. If there was a difference, then we concluded that

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carry-over toxicity had occurred because we used the same dose for a single toxicant in both

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of its exposure pulses within each experiment. The only difference was that the order was

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reversed in the second experimental group. All four experiments were analysed following the

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same method.

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Toxicokinetic-toxicodynamic modelling

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Toxicokinetics: modelling the internal dose

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The toxicokinetic model was calibrated previously using time series data of measured internal

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concentrations in pulsed exposure experiments7-9,

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concentrations in G. pulex was simulated using the previously established toxicokinetic

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models and parameters from those studies:

194

195

, ( ) ,$ ( )

14

. Here, the time-course of internal

= () × , − , () × , − ∑" , () × ! ," #

(1)

= , () × ! ," − ," () × ,"

(2),

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where Cinternal,p(t) is the concentration of the parent compound in the organism [nmol/kgw.w.],

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Cw(t) is the concentration of the parent compound in water [nmol/L], Cinternal,j(t) is the

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concentration of biotransformation product j in the organism [nmol/kgw.w.], kin,p is the uptake

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clearance coefficient [L/(kgw.w.×d)], kout,p is the elimination rate constant of the parent

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compound [1/d], kmet,j is the first-order biotransformation rate constant for the formation of

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metabolite j [1/d], and kout,j is the elimination rate constant of the biotransformation product j.

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Internal concentrations of propiconazole (PCZ) and 4,6-dinitro-o-cresol (DNOC) were

203

directly used as inputs in the toxicodynamic model; by contrast, for diazinon (Diaz) and 4-

204

nitrobenzyl chloride (NBCl), biotransformation products were the driving variables for the

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toxicodynamic model (see Tables 1 & S1, also for model parameter values). In the case of 4-

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nitrobenzyl chloride, the metabolites are unidentified and considered to act as baseline

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toxicants9. For diazinon, metabolite 1 is diazoxon, which is the toxicologically relevant


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molecule (Table 1). For propiconazole and 4,6-dinitro-o-cresol, we included only the parent

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compounds in the model, because biotransformation in G.pulex is negligible8, 14.

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Toxicodynamics: modelling survival

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We used the two limit cases stochastic death (SD) and individual tolerance (IT) of the

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General Unified Threshold model of Survival (GUTS)

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concentrations and scaled damage

9, 13

13

with explicitly modelled internal

as the dose metric. These models have been described

9, 13

214

previously

215

parameters are given in Table S2. Scaled damage is a proxy for the toxicodynamic state of

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the organism, is generally inferred by model fitting 9, 13 and it is calculated as follows:

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dDscaled (t ) = k r × (Csum tox, organism (t ) − Dscaled (t ) ) dt

218

where Dscaled(t) is the time course of the scaled damage [nmol/kgw.w.], t is time [d], Csum tox,

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organism(t)

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active chemicals in the organism [nmol/kgw.w.], and kr is the damage recovery rate constant

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[1/d]. The damage recovery rate constant captures the time course of toxicodynamics, and we

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use two different parameters, kr SD and kr IT, for the two limit cases of GUTS.

223 224

Effects of scaled damage on survival in GUTS-SD

225

The hazard rate is the probability of an organism dying at a given point in time and is

226

calculated as follows8, 13, 24:

227

%( )

, but we repeat their description here for clarity. The toxicodynamic model

(3),

is the time course of the sums of the internal concentrations of the toxicologically

= & × '() (*+, () − -, 0) + ℎ_2345367

(4),

228

where dH(t)/dt is the hazard rate [1/d], kk is the killing rate constant [kgw.w./(nmol×d)],

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Dscaled(t) is the time course of the scaled damage [nmol/kgw.w.], t is time [d], z is the threshold

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[nmol/kgw.w.], and h_controls is the background hazard rate (control mortality rate, assumed

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to be constant during the experiment, see Table S3) [1/d]. The survival probability, i.e. the

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probability of an individual surviving until time t, is given as follows:

233

S (t ) = e − H ( t )

234

where S(t) is the survival probability [unitless].

235

Effects of scaled damage on survival in GUTS-IT

(5),



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We assume a log-logistic distribution of the threshold in the study population (individual

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tolerance)8, 13. Then, the cumulative log-logistic distribution of the tolerance threshold in the

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study population, which changes over time as individuals die, is calculated as follows:

239

F (t ) =

240

where F(t) is the cumulative log-logistic distribution of the tolerance threshold over time

241

[unitless], Dscaled(t) is the time course of the scaled damage [nmol/kgw.w.], t is time [d], τ is

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time [d], α is the median of the distribution [nmol/kgw.w.], and β is the shape parameter of the

243

distribution [unitless]. Under the assumption of individual tolerance, the survival probability

244

is then given as follows 8, 13:

245

S (t ) = (1 − F (t ) ) × e − h _ controls×t

246

where S(t) is the survival probability [unitless], and h_controls is the background hazard rate,

247

i.e. the control mortality rate, which is assumed to be constant over time [1/d].

248 249

Mixture toxicity model

250

Mixture toxicity model assumptions

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Mixture toxicity models generally assume either that the toxicants act on the same target or

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on different targets 25, 26. Independent action is the model for the second assumption, which

253

applies to our combinations of toxicants. Traditional mixture toxicity models are designed for

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exposure to multiple toxicants at the same time and for constant concentrations. We expand

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the applicability domain of the mixture toxicity modelling to fluctuating, pulsed or sequential

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exposures using a dynamic model, GUTS 9, 13. As we assume independent action for

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compounds with different modes of action, we sum the hazards from the different toxicants 9,

258

1  max Dscaled (τ )   1 +  0< τ < t   α  

(6),

−β

(7),

27

, which is equivalent to multiplying the survival probabilities.

259

Mixture toxicity model equation

260

The same mixture toxicity model is used for GUTS-SD and GUTS-IT:

261

8!9 () = : ; ∑ %

262

which is equivalent to

263

8!9 () = ∏ 8 ()

(8),

(9)


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Smix (t) is the survival probability under toxic stress from i toxicants. In equation (8), we sum

265

the hazards, which is equivalent to multiplying the survival probabilities (eq. 9) and

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corresponds to the assumption that toxicants act on different target sites or via different

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toxicity pathways.

268

Treatment of NBCl metabolites in the toxicity model

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In the case of NBCl, the internal concentration of the parent compound was modelled as a

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reactive toxicant, whereas the sum of the internal concentrations of the metabolites was

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modelled as a baseline toxicant, as in a previous study 9. We used the toxicodynamic

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parameters of 1,2,3-trichlorobenzene, a well-known prototype baseline toxicant, to simulate

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the effects of the metabolites assumed to act via baseline toxicity 9. As we assumed the two

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modes of toxic action are independent, we modelled separate scaled damages for each using

275

equations (3) to (7) and then multiplied the resulting survival probabilities:

276

8,!= () = 8=+ 9, ? () × 8+,@, 9, ? () × : ;A_, +×

277 278

Results and discussion

279

Mixture toxicity model prediction

280

(10)

We compared the survival prediction determined by the mixture toxicity models with

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the observed survival (Fig. S1 to S4). The agreement or disagreement between the predicted

282

and observed survival curves after the first pulses is a measure of the inter-experimental

283

variability. In half of the cases, the predicted survival after the first pulse did not match the

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observed survival very well (Fig. S1G, S3G, S4G, H). Only in experiment two was survival

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after the first pulses predicted accurately in both groups (Fig. S2G, H); in this experiment,

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survival after the second pulses was also predicted accurately (Fig. S2G, H). The

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disagreement between the predicted and observed survival after the second pulse in

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experiment one (Diaz, Fig. S1H) or three (NBCl, Fig. S3H) is consistent with the

289

disagreement seen after the first pulse and is not necessarily an indicator that the mixture

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toxicity models fail to predict full recovery after PCZ (experiment one) or DNOC

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(experiment three). In experiment four the model predicted that all organisms would die

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almost immediately following exposure to NBCl, thus assessment of the predicted mortality

293

after DIAZ in this experiment is difficult.

294 295

We also compared the mixture toxicity model prediction with the observed survival curve in the five experimental halves where we found evidence of carry-over toxicity



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(survival after PCZ, DNOC, DNOC, DIAZ and NBCl in experiments one, two, three, four

297

and four, respectively; Fig. 2 A, C, E, G, H) and observed in four out of five cases that the

298

observed mortality following the respective second pulses exceeded the predicted mortality

299

(Fig. S6 to S10). From that observation we conclude that the toxicants interact, even if

300

exposure is days apart, and that the observed mortality following the second pulses is more

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than just continued mortality from the other toxicant of the respective first pulses in all the

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‘sequence’ treatments and the ‘inverse sequence’ treatment of experiment four (i.e. those with

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carry-over toxicity).

304

We need more studies of this type to better understand how the model performs for

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different substances and classes of chemicals and where the limits of its predictive

306

capabilities are28. Uncertainty arising from calibration experiments themselves can be

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included in the model predictions28 to better understand inter-experimental variability.

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Interesting questions arise also from the temporal mismatch in the onset of predicted and

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observed mortality (particularly NBCl, see Fig. S3 & S4) as well as our choice of toxic

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molecules (Table 1).

311

In summary, we found no evidence that the mixture models fail to predict carry-over

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toxicity or the sequence effect. However, we did find large inter-experimental variability

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evidenced by a disagreement between the predicted and observed mortalities. Since the

314

models were calibrated based on previously published experiments, this disagreement only

315

indicates that the variability in survival from one pulsed exposure experiment to the next is

316

too large to assess the performance of the model prediction. However, the experimental

317

evidence depicted in Figures S1 to S4 fully supports the sequence effect hypothesis.

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Conclusions drawn from comparisons within an experiment, such as in this study, are not

319

subject to the limitations of inter-experimental variability. Variability between experiments

320

could possibly be reduced by using laboratory-cultured organisms.

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Furthermore we identified a knowledge gap in the suite of toxicokinetic-

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toxicodynamic mixture toxicity models available. We provide equations for summing up

323

hazard rates for toxicants which are assumed to act independently. However it is also

324

conceivable to model the combined effects of toxicants that act on different targets by

325

summing up the state variable scaled damage. However there are two challenges. First,

326

summing up damage has been previously used to model combined effects of toxicants that act

327

on the same target6, 29. This raises a question: How can we reconcile that previous work with

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any future use of the same approach for toxicants acting on different targets? The task of


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reconciling this previous work with newer studies is further complicated by subtle issues and

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inconsistencies in the models used in the older studies13. Second, the model equations for

331

summing up scaled damage in GUTS-SD are straightforward to derive, however the model

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equations for summing up scaled damage in GUTS-IT are unknown and not trivial to derive.

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The sequence effect

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We observed the sequence effect in experiments one, two and three (Fig. 1). Contingency

335

table analysis of the number of dead and alive individuals at the end of each experimental

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group indicated a sequence effect for the combinations of DIAZ and PCZ (p

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